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Bernard-Soulier syndrome (BSS) is a rare severe autosomal recessive bleeding disorder. To date heterozygous carriers of BSS mutations have not been shown to have bleeding symptoms. We assessed bleeding using a semi-quantitative questionnaire, platelet parameters, PFA-100 closure times, ristocetin response, GP Ib/IX expression and VWF antigen in 14 BSS patients, 30 heterozygote carriers for related mutations and 29 controls. Eight mutations in GP1BA, GP1BB or GP9 were identified including four previously unknown pathogenic mutations. Subjects with BSS reported markedly more mucocutaneous bleeding than controls. Increased bleeding was also observed in heterozygotes. Compared to controls, patients with BSS had lower optical platelet counts (P < 0.001), CD61-platelet counts (P < 0.001) and higher mean platelet volume (17.7 vs. 7.8 fL, P < 0.001) and ristocetin response and closure times were unmeasurable. Heterozygotes had higher MPV (9.7 fL, P < 0.001) and lower platelet counts (P < 0.001) than controls but response to ristocetin and closure times were normal. The VWF was elevated in both BSS and in heterozygotes (P = 0.005). We conclude that heterozygotes for BSS mutations have lower platelet counts than controls and show a bleeding phenotype albeit much milder than in BSS. Both patients with BSS and heterozygote carriers of pathogenic mutations have raised VWF., (© 2014 Wiley Periodicals, Inc.)

To assess sources of variability in platelet function tests in normal subjects, 64 healthy young adults were tested on 2-6 occasions at 2 week intervals using four methods: platelet aggregation (AGG) in platelet-rich plasma (PRP) in the Bio/Data PAP-4 Aggregometer (BD) and Chrono-Log Lumi-Aggregometer (CL); and AGG in whole blood (WB) in the CL and Multiplate Platelet Function Analyser (MP), with ATP release (REL) in CL-PRP and CL-WB. Food and medication exposures were recorded prospectively for 2 weeks prior to each blood draw. At least one AGG abnormality was seen in 21% of 81 drug-free specimens with CL-PRP, 15% with CL-WB, 13% with BD-PRP and 6% with MP-WB, increasing with inclusion of REL to 28% for CL-PRP and 30% for CL-WB. Epinephrine AGG and REL were significantly reduced in males (P < 0·0001). Ristocetin AGG and collagen and thrombin REL were significantly reduced in Blacks (P < 0·0001). One-third of specimens drawn following flavonoid-rich food exposures had aberrant results, compared to 8·5% of specimens without such exposures (P = 0·0035). PRP tests had less intra-individual variation than WB tests. Gender, race, diet and test system affected results of platelet function testing in healthy subjects, suggesting caution when interpreting the results of platelet function testing in patients., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

It has been shown that β(2) -glycoprotein I (β(2) GPI) interacts with von Willebrand factor (VWF) in a glycoprotein (GP)Ib binding state. Given the presence of active VWF multimers in thrombotic thrombocytopenic purpura (TTP), we speculated that β(2) GPI might play a role in TTP. We found that β(2) GPI plasma levels were significantly lower in acute and remission TTP patients than in normal controls, showing a direct correlation with ADAMTS 13 levels and an inverse correlation with the extent of VWF activation. In vitro flow experiments demonstrated that β(2) GPI can block platelet adhesion to endothelial cell-derived VWF strings. We confirmed the direct binding of β(2) GPI to VWF by surface plasmon resonance, and determined that domain I of β(2) GPI is the binding site of VWF A1 domain. Adhesion of β(2) GPI to erythrocytes and platelets was increased in the presence of active VWF, indicating that β(2) GPI may be cleared from the circulation during TTP episodes together with blood cells. Our findings suggest that β(2) GPI may protect from the effects of hyper-functional VWF by inhibiting its interaction with platelets., (© 2012 Blackwell Publishing Ltd.)

Acquired loss of functional von Willebrand factor (VWF) has been termed the acquired von Willebrand syndrome (AVWS). AVWS is a rare adult-onset bleeding diathesis that is clinically similar to congenital von Willebrand disease (VWD), and occurs with a variety of autoimmune, lymphoproliferative, or myeloproliferative disorders. We have identified four patients with AVWS in association with immunoglobulin light chain (AL) amyloidosis. These patients, lacking any pre-existing or family history of abnormal bleeding, developed cutaneous, mucosal, or gastrointestinal bleeding in the course of their disease without deficiency of clotting factor X or other factors; the activated partial thromboplastin time (aPTT) was prolonged in three out of the four cases. Despite normal VWF antigen levels, VWF ristocetin cofactor activity (VWF:RCo) was low. Electrophoresis patterns of high molecular weight (HMW) VWF multimers were abnormal in two of the four cases. Two of the patients were treated with high-dose intravenous melphalan followed by autologous stem cell transplantation (HDM/SCT) and achieved hematologic remission. In these two patients, the bleeding diathesis improved and the coagulation parameters normalized, confirming a causal relationship between the plasma cell dyscrasia and the AVWS. AVWS should be considered in AL amyloidosis patients with hemorrhagic diatheses and normal clotting factor levels., ((c) 2007 Wiley-Liss, Inc.)

Type 2B von Willebrand disease (VWD) is characterised by an increased affinity of von Willebrand factor (VWF) for its platelet receptor glycoprotein Ib (GPIb). This feature is usually studied in vitro by a ristocetin-dependent VWF platelet-binding assay, which has some limitations as it requires [e.g. (radio)-labelled anti-VWF antibodies and normal formaldehyde-fixed platelets]. We, here, extended the applicability of an enzyme-linked immunosorbent assay-based method previously described for the measurement of ristocetin co-factor activity that used a recombinant fragment of GPIb (rfGPIb alpha) and horseradish peroxidase-labelled rabbit anti-human VWF antibodies for measuring the captured ristocetin-VWF complexes on the rfGPIb alpha. Thirty-one type 2B VWD patients from 15 families with eight different known mutations were studied. VWF in plasma from 28 of these patients bound better than normal VWF at 0.2 mg/ml ristocetin, with the ratio, optical density (OD) patient/OD normal pool plasma, higher than 1.8. For two of the three other patients with no enhanced response of plasma VWF, the platelet lysate VWF showed an enhanced binding capacity; for the last patient, the results in other members of the family are unequivocal. We conclude that, this new method for measurement of plasma or platelet VWF-binding capacity offers great advantages for correct type 2B VWD diagnosis.

von Willebrand disease (VWD), the most common inherited bleeding disorder, is very heterogeneous, both in its phenotype and genotype. One particular molecular mechanism of VWD is due to recombination events between the true gene and its pseudogene on chromosome 22. We assessed the frequency and extension of such events in 50 multi-ethnic index patients with severe VWD type 3 and in five index patients with VWD type 2M Vicenza. One additional unclassified patient had been diagnosed with possible VWD in Russia solely on a clinical basis. Gene conversions, previously thought to be rare events, were identified in >10% of our study population: in six multi-ethnic patients with severe VWD type 3, in one patient with VWD type 2M Vicenza and the Russian patient was finally diagnosed with VWD type 2B New York/Malmoe. Our results suggest a significant contribution of this particular molecular mechanism to the manifestation of VWD. The location of the gene conversions, their extension and their occurrence as homozygous, compound heterozygous or heterozygous mutations determines the resulting phenotype.

Interaction of platelet glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) is essential for thrombus formation, particularly under high shear conditions. Previous case-control studies indicated that two GPIb alpha polymorphisms, (145)Thr/Met and/or variable number (1-4) tandem repeats of 13 amino-acid sequences, are associated with arterial thrombosis. The (145)Met-allele and the 3R- or 4R-allele is associated with increased risk. However, there is little clear experimental data to support this association. To elucidate the functional effects of these polymorphisms, we prepared recombinant GPIb alpha fragments and tested them in vitro. The dissociation constants of ristocetin-induced (125)I-labelled VWF binding to two forms of soluble recombinant GPIb alpha [(1)His-(302)Ala, either (145)Thr (145T) or (145)Met (145M)] were not different. Four types of Chinese hamster ovary cells expressing full-length GPIb alpha beta/IX, 145T with one repeat (T1R), 145M with one repeat (M1R), 145T with four repeats (T4R), and 145M with four repeats (M4R), were prepared, and cell interactions with immobilized-VWF were examined under various shear conditions. The cell rolling velocity of M4R under a shear condition of 114/s was significantly slower than that of T1R. Intermediate values were obtained with M1R and T4R. The results suggest that M4R interacts more strongly with VWF under flow conditions.

The haemostatic activity of von Willebrand Factor (VWF) is dependent on VWF multimer stability. Fragments, arising from the proteolytic cleavage of the multimers by VWF-cleaving protease, are ineffective in initiating platelet aggregation. We designed a simple method to determine the protease activity, which was expressed as the reduction in the level of ristocetin-induced platelet aggregation between the inactive enzyme and the enzyme when activated by the addition of calcium. Samples from senior women (n = 14) with chronic coronary heart disease (CHD), age-matched control subjects (n = 15) and young healthy individuals (n = 13), as well as patients with either thrombotic thrombocytopenic purpura (TTP) (n = 2) or von Willebrand disease type 2A (VWD-2A) (n = 2), were examined. The lower protease activity observed in the CHD group, compared with the control subjects, indicated that the increased levels of VWF found in CHD relate to impaired enzyme function. The assessment of TTP patients reconfirmed the reduced protease activity previously observed in this disorder. In VWD-2A, normal enzyme function was observed, suggesting that there is an increased sensitivity of the mutated VWF protein to the protease, rather than an increase in the activity or quantity of the enzyme involved in pathogenesis. In summary, the present simplified method efficiently determines VWF protease activity and is suitable for use in laboratories where platelet aggregation analyses can be performed.

We have designed a simple test for the early diagnosis and treatment monitoring of thrombotic thrombocytopenic purpura (TTP). We examined plasma from 24 TTP patients and normal plasma using a cone and plate(let) analyser (CPA). Test plasma was mixed with citrated normal whole blood (group O) and subjected to flow at a shear rate of 1800/s. Mixing normal plasma (12.5, 25, 50 or 75 microl) with heterologous normal whole blood (final volume of 200 microl) resulted in a decrease of surface coverage (SC, maximally by 63%) and, to a lesser extent, of average size (AS, maximally by 37%) due to dilution of the blood sample. In contrast, mixing the same quantities of acute TTP plasma with normal blood yielded an increase in both SC (up to 125%) and AS (up to 130%). Increased SC and/or AS were detected in all 15 patients in acute phase and in three out of 14 patients in remission. Following repeated plasmapheresis, the enhanced platelet deposition in five patients with acute TTP returned to almost normal patterns. Mixing plasma from patients with other thrombocytopenic conditions in this way resulted in a decrease in both SC and AS, and did not differ from control subjects. In conclusion, the CPA is a simple and specific laboratory test that can be used for the diagnosis and monitoring of plasma exchange therapy in TTP.

Diagnosis of von Willebrand disease (vWD) is based on a panel of laboratory tests that measure the amount and function of von Willebrand factor (vWF). In population studies, vWF is higher in African Americans than Caucasians. Bleeding time, factor VIII activity (FVIII), vWF antigen (vWF:Ag), "vWF activity" ELISA (vWF:Act), ristocetin cofactor (vWF:RCof), and ristocetin-induced platelet aggregation (RIPA) were measured on 123 women with menorrhagia and 123 randomly selected control women; 70 cases and 76 controls were African American. Among controls, African Americans had significantly higher levels of vWF:Ag (mean 120 vs. 102 U/dl, P = 0.017). Among all subjects, African Americans had higher levels of vWF:Ag (mean 123 vs. 103, P = 0.001), vWF:Act (mean 101 vs. 89, P = 0.006), and FVIII (mean 118 vs. 104, P = 0.008). VWF:RCof did not differ between races (93 vs. 94 U/dl). RIPA was reduced in African Americans (P < 0.0001). In both races, women with type O blood differed significantly from those with other ABO types in vWF:Ag, vWF:Act, FVIII, and vWF:RCof. Based on criteria of two or more tests below race- and ABO-specific reference ranges, 6.5% of menorrhagia cases and 0.8% of controls were classified as having vWD, or its phenocopy. Among Caucasians, no controls and 7 cases (15.6%) were classified as affected, and in African Americans, 1 control (1.3%) and 1 case (1.4%) were so classified. Racial differences in vWF further complicate the issues surrounding diagnosis of vWD. The finding of increased vWF:Ag not accompanied by increased vWF:RCof has implications for understanding the structure-function relationships of vWF. Published 2001 Wiley-Liss, Inc.

A large variety of platelet dysfunctions has been described in chronic myeloproliferative disorders. These abnormalities may be due to deficiency of platelet granules, arachidonic acid metabolism defects or platelet membrane glycoproteins abnormalities. In this study we intend to detect the incidence of platelet function defects in 76 patients with various types of chronic myeloproliferative disorders. The platelet activity was studied in vitro by measuring platelet aggregation in response to ADP, epinephrine, collagen, arachidonic acid and ristocetin. These results were subsequently correlated with bleeding time and clinical aspects (bleeding or thrombosis). We found complex changes in platelet response with all agonists, in varied proportions. These abnormalities include absent, decreased or abnormal platelet aggregation response. In a few cases we found a markedly decreased, almost absent platelet response to all agonists while in some patients a normal platelet aggregation was noted. The correlation between these results and template bleeding time, thrombotic or hemorrhagic events and the type of diseases was difficult to establish and sometimes conflictual. Despite this fact, we consider that investigating platelet aggregation may be useful not only for the assessment of the hemostatic balance in chronic myeloproliferative disorders but also for a better insight into cell abnormalities occurring in these pathologic conditions.

A recent development in antithrombotic research allows the inhibition of platelet aggregation via protection of the glycoprotein IIb/IIIa receptor on the platelet membrane. We hypothesized that a GP IIb/IIIa receptor inhibitor would inhibit thromboxane-induced platelet aggregation during circulation in our in vitro ventricular assist device (VAD) circuit and preserve long-term platelet function. Twenty-one in vitro nonpulsatile centrifugal VAD circuits were simulated for 4 days using 450 ml of fresh human whole blood with or without glycoprotein IIb/IIIa receptor inhibitor (tirofiban). Platelet aggregation and degranulation were measured in whole blood induced by ristocetin, collagen, ADP, and thromboxane A2 (TXA2). The tirofiban-treated group preserved the platelet count and tended to exert these beneficial effects by inhibiting pathologic platelet aggregation induced by TXA2, collagen, and ADP as well as degranulation. Tirofiban may be useful in preserving platelet number and function during clinical VAD use.

Extensive microvascular platelet aggregation is characteristic of thrombotic thrombocytopenic purpura (TTP). Previous studies have indicated that abnormalities of von Willebrand factor (vWf) are often present in TTP patient plasma. There has not been previously any direct evidence linking these abnormalities to the process of intravascular platelet aggregation in TTP. We used flow cytometry to analyze the binding of vWf to single platelets, and the presence of platelet aggregates, in the blood of 4 children with chronic relapsing (CR) TTP and 5 adults with single episode or recurrent TTP. vWf on the single platelets of CRTTP patients at all time points studied was significantly increased compared to controls, and was increased further as platelet counts decreased to levels below 40,000/microl. The single episode and recurrent adult TTP patients had platelet aggregates in the blood, as well as increased vWf on single platelets, before therapy commenced and thereafter until recovery was in process. In the one unresponsive single episode TTP patient, vWf on single platelets remained elevated, and platelet aggregates persisted, until her death. The platelet alpha-granular protein, P-selectin, was not increased on the single platelets of most TTP blood samples, suggesting that it is vWf from plasma (rather than from alpha-granules) that attaches to platelet surfaces in association with platelet aggregation. These results suggest that vWf-platelet interactions are involved in the platelet clumping process that characterizes TTP.

Nine patients (10 infusions) with a confirmed diagnosis of type 3 VWD were infused with von Willebrand factor (human), a preparation of von Willebrand factor (VWF) with a very low factor VIII content. Each patient was infused with one dose of approximately 50 or 100 iu ristocetin cofactor activity (VWF:RiCoF) per kg body weight. Bleeding times were performed during the 24 h period after infusion. Plasma samples were obtained over the 96 h period after infusion and were analysed for factor VIII coagulant activity (FVIIIC), VWF:RiCoF, von Willebrand factor antigen (VWF:Ag), and multimers. The FVIIIC data were analysed by non-linear least-squares analysis assuming constant FVIIIC 'synthesis' and exponential decay. The VWF data were fitted for exponential decay. The average decay rates for FVIIIC, VWF:RiCoF and VWF:Ag were 0.041, 0.061 and 0.056 respectively. The average calculated 'synthesis' rate for FVIIIC was 6.4 u/dl/h. The synthesis of FVIIIC was slightly faster and the decay slightly slower following the infusion of 100 iu VWF:RiCoF/kg than of 50 iu VWF:RiCoF/kg. Correction of the bleeding time was strongly dose dependent. At 4 h post infusion the median bleeding time was 9 min following a dose of 50 iu VWF:RiCoF/kg versus 3 min with a dose of 100 iu VWF:RiCoF/kg. There was no decrease in the bleeding time until the level of VWF:Ag or VWF:RiCoF reached > 100 u/dl.

Type 2B von Willebrand disease is characterized by an abnormal von Willebrand factor molecule with increased affinity for the platelet glycoprotein (GP) Ib-IX receptor. A diagnostic feature of type 2B von Willebrand disease is a characteristic loss of von Willebrand factor high molecular weight multimers. In vitro, the soluble interaction of normal von Willebrand factor with platelets can be initiated with exogenous modulators, the most common being the antibiotic ristocetin. The variant molecules resulting in type 2B von Willebrand disease can sustain binding to platelets at subnormal levels of ristocetin. We characterized the von Willebrand factor gene of an individual with type 2B von Willebrand disease and identified a nucleotide transition resulting in an Arg543-->Trp amino-acid substitution within the GP Ib-IX binding domain of von Willebrand factor. In this study we demonstrate that a recombinant plasmid capable of expressing the isolated GP Ib-IX binding domain of von Willebrand factor, and containing the Arg543-->Trp amino-acid substitution, secretes a dimeric molecule that supports platelet agglutination using subnormal levels of ristocetin. These results demonstrate that the mutation at position 543 increases the affinity between the variant molecule and platelet GP Ib-IX as an intrinsic feature of the isolated von Willebrand factor domain. Thus, structural perturbations within the GP Ib-IX binding domain that are independent of the von Willebrand factor multimeric structure can sufficiently increase the affinity of von Willebrand factor to sustain platelet aggregation, using subnormal levels of ristocetin.

P-selectin (also called CD62, GMP-140, PADGEM, CD62P) is a recently described member of a family of vascular adhesion receptors expressed by activated platelets and endothelial cells that are involved in leucocyte cell adhesion. The aim of this study was to characterize a new monoclonal antibody (LYP7) directed against activated human blood platelets that inhibits ristocetin-induced platelet aggregation. Immunoadsorbent affinity chromatography and immunoprecipitation studies showed that LYP7 (IgG1) bound a surface-labelled glycoprotein (GP) which changed its apparent molecular mass (M(r)) on reduction from 138 kD (situated below GPIIb) to 148 kD (above GPIIb alpha). LYP7 and S12, a monoclonal antibody directed against P-selectin immunoprecipitated the same band. Using ELISA assay, purified P-selectin was shown to bind LYP7 and S12 monoclonal antibodies. Binding sites of 125I-labelled LYP7, which was greatly increased on thrombin-stimulated (2 U/ml) washed platelets (10825 +/- 2886, mean +/- SD) Kd = 1.5 +/- 0.5 nM) compared to resting platelets (2801 +/- 1278, mean +/- SD) (Kd = 1.5 +/- 0.6 nM), was found to be normal on thrombin-stimulated platelets taken from a patient with grey platelet syndrome or a patient with Glanzmann thrombasthenia. LYP7 (IgG1, F(ab')2 or Fab fragments) inhibited ristocetin-induced platelet aggregation of platelets in a dose-dependent fashion without affecting the binding of von Willebrand (vWf) factor. However, agglutination of formaldehyde-fixed platelets induced by ristocetin was not affected by monoclonal antibody LYP7. In addition, the binding of thrombin-activated platelets to neutrophils was inhibited by monoclonal antibody LYP7. These results strongly suggest that P-selectin, by promoting cell-cell contact, may play an active role in platelet-platelet interactions.

Allergic vasculitis phenomena seem to be involved in Henoch-Schönlein purpura (HSP). Elevated plasma levels of von Willebrand factor (vWf) are a well recognized feature of vasculitis and have been taken as an indication of in vivo endothelial cell damage. Plasma factor VIII:C and vWf levels and vWf multimeric pattern were studied in 8 patients with HSP, during active disease and twice during the remission (3 and 9 months later). Plasma vWf multimeric composition was evaluated using low resolution gels which better resolve large vWf multimers. During active disease plasma factor VIII:C, vWf:Ag, and vWf:RCoF were normal in 5% of patients and increased in three, but in each patient, platelets appeared to aggregate at doses of ristocetin lower than in normals. Furthermore, all patients demonstrated the presence of abnormally large vWf multimers usually found only in platelets and endothelial cells. Three and 9 months later, during remission, in spite of the normalization of factor VIII:C and vWf levels, the abnormal multimers were still detectable, as well as hyper-responsiveness to ristocetin. These findings confirm that damage and/or perturbation of endothelial cells is associated with HSP. Moreover, the persistence of abnormality in the vWf multimeric pattern, when the disease is inactive, suggests that the mechanisms involved operate through the entire clinical course.

Type 2B von Willebrand disease (vWD) is characterized by an increased affinity of von Willebrand factor (vWF) for binding to platelet glycoprotein Ib (GpIb). Most type 2B candidate mutations are clustered in the 509-695 disulphide loop but three of them (H505D, L697V and A698V) are outside this loop. We confirm here that the A698V mutation is a type 2B mutation by its expression in Cos-7 cells. As the L697V and A698V type 2B mutations both induce the presence of a valine residue in the 694-708 sequence, we created and expressed different mutated recombinant vWFs (rvWFs), in substituting the other leucine and alanine residues of this sequence (at positions 694, 701 and 706) into valine resides. V694rvWF and V706rvWF displayed decreased ristocetin-induced GpIb binding showing that it is not always the presence of a valine residue that may explain the increased affinity of type 2B vWF for GpIb. We also compared the interaction with platelets of V697rvWF and V698rvWF to those obtained with rvWFs reproducing two prevalent type 2B mutations located in the loop (R543W and V553M). We show that the two mutations located in the loop are more reactive than the two mutations identified outside the loop.

Mutual interactions between neutrophils (PMN) and platelets are recognized to be important in modulating the respective functions of these two cell types. Here we show that primary granule secretion from appropriately-stimulated PMN can lead to complete proteolytic removal of GPIb from the platelet surface. Thus, when the PMN in PMN/platelet mixtures were stimulated by FMLP, platelets lost GPIb as measured by ristocetin-induced aggregation, flow cytometry and SDS-PAGE analysis. This loss was most marked when PMN were primed by GM-CSF, and could be inhibited by a specific elastase inhibitor. As expected, the alpha 1-antiproteinase in plasma inhibited GPIb loss, but when PMN were strongly stimulated by FMLP and GM-CSF in the presence of platelets this inhibition was incomplete or absent. It is concluded that joint priming of PMN with GM-CSF and platelets can cause a previously unrecognized degree of primary granule secretion which, via elastase, leads to platelet GPIb loss. We suggest that this loss is likely to be of physiological and pathophysiological importance.

A new variant of von Willebrand's disease has been discovered in 2 members of a Macedonian family of 6. The proband, an 8-year-old boy, showed a prolonged bleeding episode on 1 occasion. Ristocetin-induced platelet aggregation and bleeding time were normal. In plasma, ristocetin cofactor activity (RCo) and von Willebrand factor (vWf) antigen were reduced to the same clearly low level. The determination of vWf antigen of platelets resulted in borderline values, while RCo was clearly reduced. Low- and intermediate-resolution agarose gel electrophoresis showed absence of the largest multimers in plasma vWf, and slight reduction in platelet vWf. High-resolution gels revealed abnormal multimeric structure only in plasma vWf. The smaller multimers could be resolved in a broad central band flanked by 4 fainter satellite bands; however, satellite bands close to the central band were more intense, and more distant ones were fainter, compared to normal plasma. The central band of the fastest-moving multimer was markedly intensified, and the mobility of the whole quintuplet was slightly reduced. Heredity seems to be autosomal-dominant. No mutation was found in exon 28 of the vWf gene. Because there was only 1 mild bleeding episode in the family, this structural variation seems to have only little clinical consequence. We conclude that this vWf abnormality is different from those observed in other type II variants previously described. Based on the revised classification by the International Society on Thrombosis and Haemostasis, we proposed designation type 2A-Bern for this new subtype.

A decrease of platelet agglutination induced by ristocetin has been described in cirrhotic patients. In order to investigate the relationship of such phenomenon with a putative defect on platelet membrane glycoprotein Ib, we have studied the quantitative and functional status of Gp Ib in eleven severe alcoholic cirrhotic patients, and the ability of their formalin-fixed platelets to agglutinate in the presence of normal plasma plus ristocetin. Interestingly, we found a significant decrease of immunoreactive GP Ib molecules (9,978 +/- 1,534 vs. 17,064 +/- 404 molecules per platelet) and ristocetin-dependent binding of vWF (9,113 +/- 1,338 vs. 13,992 +/- 1,968 molecules per platelet) (P < 0.01) in comparison to the levels found in a control group of healthy subjects. Immunoblotting analysis of platelet lysates confirmed the reduction of GP Ib level in cirrhotic patients, but showed no modification on the precipitation pattern of this glycoprotein. The ristocetin-induced platelet agglutination (light transmission %) was also significantly lower in patients with cirrhosis (48 +/- 7.6 vs. 92 +/- 3.6, P < 0.01), and correlated with the binding of normal vWF (r = 0.863, P = 0.0013). Patients with bleeding times longer than 7 min and/or clinical history of bleeding episodes showed the lowest values of platelet agglutination and GP Ib level. In conclusion, our present results indicate that liver cirrhosis is associated with a relevant decrease of functional GP Ib molecules on the platelet surface. This reduction might be related to the prolongation of bleeding time and to the bleeding diathesis observed in cirrhotic patients.

Thrombin induces a redistribution of glycoprotein (GP) Ib/GP IX complex from the platelet surface into the surface connected canalicular system (SCCS). This redistribution results in a reduced interaction of platelet GP Ib with von Willebrand factor (vWF) bound to subendothelium leading to impaired platelet adhesion. In this study we show that the platelet aggregation and degranulation require concentrations of thrombin above 0.05 U/ml, while the decrease in GP Ib function (about 50% of control value), as determined by ristocetin induced platelet agglutination, can be induced by lower concentrations (0.01-0.04 U/ml). Moreover, we show that when adding thrombin inhibitors to the platelets preincubated with < 0.04 U/ml thrombin for 5 min, their agglutinability by ristocetin was gradually recovered within 30 min, indicating that in these conditions the decrease in platelet adhesiveness is reversible. Immuno-electromicroscopic study showed that this restoration of platelet GP Ib function was associated with a reversed translocation of GP Ib from the SCCS to the plasma membrane. The data obtained from counting gold particles showed that the ratio of GP Ib immunolabelling on the external membrane versus that on the SCCS was 3.31 +/- 0.90 for resting platelets, down-regulated to 0.84 +/- 0.13 (P < 0.05 versus resting platelets) for the platelets treated with 0.04 U/ml thrombin and returned to 2.63 +/- 2.21 (P > 0.05 versus resting platelets) after incubation for 30 min with hirudin. However, the translocation of GP Ib was poorly reversed by thrombin inhibitors when higher concentrations of thrombin were used which induced platelet aggregation and large extent of degranulation. We conclude that thrombin affects platelets in a dose dependent manner, and that at low concentrations the decrease in platelet GP Ib related function is a reversible phenomenon.

Thrombocytopenia after desmopressin (DDAVP) infusion is usually observed in patients with type IIB von Willebrand disease (vWD). No other subtypes of vWD with thrombocytopenia after DDAVP have been reported so far. We describe here the occurrence of thrombocytopenia after DDAVP in a 39 year old male and his son with phenotypic characteristics of type I vWD, "platelet discordant subtype." After DDAVP, the abnormal ristocetin cofactor/von Willebrand factor antigen ratio in plasma was not corrected and the bleeding time remained markedly prolonged. Platelet count dropped 30 min after DDAVP (from 279 to 96 x 10(3)/microL in the propositus and from 298 to 116 x 10(3)/microL in his affected son) and returned to normal at 60 min. Platelet clumping was evident on peripheral blood smears obtained after infusion. These cases indicate that after DDAVP thrombocytopenia can occur in vWD other than type IIB.

Binding of different antibodies to the GPIIb-IIIa complex in resting (AP2, EDU3, C17) or activated platelets (PAC1) was studied by flow cytometry in a patient with a platelet defect involving GPIIb-IIIa related functions. The patient has a mild history of bleeding. Aggregation induced by ADP and collagen were absent but normal response was obtained with ristocetin. Platelets from the patient do not bind fibrinogen. Perfusion studies with flowing blood showed that patient's platelets have a marked impairment in the process of spreading and aggregate formation on vascular subendothelium. Electrophoretic studies in SDS-polyacrylamide gels demonstrated the presence of normal amounts and normal mobility of GPIIb-IIIa. Fibrinogen was present in the patient's platelets (68-74% of controls). The binding of AP2 and EDU3 to patient's resting platelets was normal as assessed by flow cytometry. In contrast, a decreased presence of the C17 antigen (10 fold lower than control platelets) was detected in resting platelets and a markedly reduced binding of PAC1 was found in thrombin activated platelets. These studies suggest that C17 recognizes an epitope of the GPIIb-IIIa in resting platelets that is implicated in the regulation of adhesive and cohesive properties of GPIIb-IIIa. Studies on this patient might be helpful for the understanding of GPIIb-IIIa functions.

We have identified a patient with von Willebrand's disease (vWD) resembling type IIB vWD, with increased ristocetin induced platelet aggregation (RIPA), the absence of the large multimers of von Willebrand factor (vWF) in plasma, and the presence of the large multimers in platelets in whom a family study indicated a probable double heterozygous inheritance pattern. The propositus was a 12-year-old boy with frequent epistaxis and bruising. Abnormal hemostatic findings included a prolonged bleeding time (BT), decreased levels of factor VIII coagulant activity (VIIIC), von Willebrand factor antigen (vWF:Ag), ristocetin cofactor (RCof), and an increased RIPA. In the presence of ristocetin, binding of the patient's plasma vWF to normal platelets was increased but binding of normal vWF to his platelets was normal. SDS-agarose gel (1.5%) electrophoresis revealed that plasma vWF lacked the large multimers, and 3.0% gel electrophoresis revealed that the multimers had a 5-band pattern similar to normal. The above findings were consistent with type IIB vWD, but 1-deamino[8-D-arginine]-vasopressin (DDAVP) infusion resulted in a shortened BT and the transient appearance of large multimers without a decrease in the platelet count. Family studies revealed that his mother has mild bleeding symptoms, decreased VIIIC, vWF:Ag, and RCof levels and normal to slightly reduced RIPA with a multimer pattern consistent with type I vWD. In contrast, the father, sister, and paternal grandfather were asymptomatic, with a slightly decreased VIIIC level but a normal BT and vWF:Ag and RCof levels. Their RIPA and vWF binding to normal platelets were increased, but unlike the propositus their plasma contained large multimers. We concluded that the propositus is a type IIB-like variant differing from previously reported IIB variants in two ways: 1) his response to DDAVP and 2) a possible double heterozygous mode of inheritance rather than the usual dominant route.

Platelets show a rapid reduction in their responsiveness to aggregating agents during storage for transfusion, but little is known about reversal of this defect in vivo after transfusion. In this study, fresh and stored platelets from the same donor (n = 12) were labelled with 111In or 51Cr, respectively, mixed, and simultaneously infused. Blood samples were taken for up to 5 d post-infusion, and the functional behaviour of the labelled platelets ex vivo was measured by retention on glass bead columns, and by whole blood aggregability to ADP, epinephrine and ristocetin. Aggregation was determined by filtering aggregated samples through a column of cotton wool to remove the aggregates, and quantitated as per cent decrease in radioactive counts. The study showed that, although infused radiolabelled 5 d stored platelets had a significantly lower aggregability towards ADP and epinephrine immediately (1 h) after infusion (32% and 29%, respectively, of fresh platelet values), a complete restoration to fresh platelet levels was found 24-72 h post-infusion, with no further change observed over the ensuing 5 d with either fresh or stored labelled platelets. A slightly (6-9%) lower adhesion to both uncoated and collagen-coated beads was found for the stored platelets throughout the 5 d period of study post-infusion. In conclusion, these studies show that, with ex vivo testing, platelets stored for 5 d quickly recovered adhesion and aggregability capabilities similar to that of fresh platelets, suggesting that the functional lesion developed during storage is quickly and completely reversed after infusion.

Patients with uraemia have abnormal platelet function that may be partially corrected by haemodialysis, cryoprecipitate or 1-desamino-8-D-arginine vasopressin (DDAVP). We studied the platelet von Willebrand factor receptor, glycoprotein Ib (GPIb), and plasma von Willebrand factor (vWF) in uraemic patients undergoing chronic haemodialysis. Using the slope of agglutination of formalin-fixed platelets as an index of response to ristocetin (with a constant amount of normal plasma as a source of vWF), we found the response of platelets from uraemic patients, both before (2.7 +/- 1.5, n = 40) and after dialysis (1.2 +/- 1.2, n = 40) to be significantly less than that for normal controls (14.1 +/- 10.2, n = 20; P less than 0.001). In addition, the agglutination response of platelets obtained after dialysis was less than that of platelets obtained before dialysis (P less than 0.001). Immunoblotting demonstrated decreased or absent staining of glycocalicin, a subunit of GPIb, in platelet lysates from 25 patients. All platelet samples with reduced glycocalicin also had decreased responses to ristocetin. Tritium-labelled platelets from seven patients showed decreased labelling of a protein with an electrophoretic mobility equivalent to that of GPIb (140,000 daltons). In addition, platelets with the lowest levels of surface GPIb, as demonstrated by flow cytometry, also had decreased ristocetin agglutination and decreased staining on immunoblot. Levels of von Willebrand factor antigen and ristocetin cofactor in plasma from 10 patients were generally within the normal range, although postdialysis levels tended to be higher than pre-dialysis levels. The pre- and post-dialysis plasma vWF multimeric patterns were normal.

We have previously observed calpain activity (calcium-dependent cysteine protease) in sera from patients with acute thrombotic thrombocytopenic purpura (TTP). The calpain activity was not present following recovery and was not detected in other thrombocytopenic disorders. We postulated that this enzyme could participate in the pathogenesis of TTP. Because other investigators have demonstrated abnormalities of von Willebrand factor (vWF) in patients with TTP, we proposed that calpain might interact with vWF in TTP. To challenge this hypothesis, we measured the binding of untreated and calpain-treated vWF to normal and ADP or calpain activated platelets. Untreated vWF bound in a specific and saturable fashion to activated platelets, but only at low (30 microM) calcium concentrations. Von Willebrand factor did not bind to activated platelets at physiological (2 mM) calcium concentrations. Calpain proteolysis of vWF changed the binding characteristics of the vWF so that it had greatly increased binding to both ADP and calpain activated platelets. The calpain-proteolyzed vWF bound to activated platelets at both low and physiological calcium concentrations, and was capable of causing platelet aggregation. The calpain-proteolyzed vWF bound to the activated platelets via glycoproteins IIb/IIIa as demonstrated by inhibition studies using monoclonal antibodies against glycoproteins IIb/IIIa and Ib. It also had a high binding affinity and was capable of inhibiting the binding of radiolabelled fibrinogen to the activated platelets at physiological calcium concentrations. Calpain also proteolyzed fibrinogen, but the calpain altered fibrinogen had normal platelet reactivity. These studies provide further insight into the pathogenesis of the platelet aggregation of thrombotic thrombocytopenic purpura. Calpain proteolyses vWF and can produce the characteristic loss of large multimers seen on sodium dodecyl sulphate (SDS)-agarose gel electrophoresis. The altered vWF is highly reactive with activated platelets and binds to platelet glycoproteins IIb/IIIa and participates in formation of the platelet aggregates that characterize this disease.

Platelets from patients with platelet-type von Willebrand disease (vWD) were used as immunogens for the production of murine monoclonal antibodies (MoAbs). One such MoAb, C-34, inhibited ristocetin-induced aggregation of patient or normal platelets, but not aggregation induced by other aggregating agents. As demonstrated by crossed-immunoelectrophoresis, C-34 recognized an epitope within the GPIb/IX complex. In indirect immunofluorescence studies on fresh platelets, the ratio of any of four different anti-GPIb MoAbs to one another was near unity (0.88-1.14) both for normals and for patients. In contrast, the ratio of the binding of C-34 to such a MoAb (AP-1) was 0.31 +/- 0.02 (means +/- SE) for normal platelets and significantly increased to 0.54 +/- 0.01 for patient platelets (P less than 0.001). In NP-40 lysates of 3H-labelled platelets, saturating concentrations of C-34 produced much fainter bands than did AS-2 or other anti-GPIb MoAbs in the GPIb and GPIX regions. In contrast to the other anti-GPIb MoAbs, C-34 did not bind to the purified 125I-labelled glycocalicin fragment of GPIb, to the glycocalicin derivative identified by crossed-immunoelectrophoresis, or to the amino-terminal approximately 40 kDa portion of GPIb alpha cleaved from 3H-labelled platelets by trypsin. C-34 appears to be the first MoAb that is quantitatively informative in identifying the abnormal platelets in platelet-type vWD. The observed differences between the patient and normal platelets may reflect an abnormality in the primary structure of the GPIb/IX complex. Alternatively, patient platelets may have an abnormality of other structures near this region that impose less of a steric hindrance upon binding of antibody to the C-34 epitope.

We have studied the ability of the plasma to induce aggregation of both homologous and heterologous platelets in four patients with hemolytic-uremic syndrome (HUS) associated with chemotherapy with mitomycin C (MMC). Neither platelet aggregation was elicited by patients' plasmas nor the in vitro addition of purified von Willebrand factor (vWF) had any effect on the aggregation pattern. In addition, ristocetin-induced binding of patients' vWF to formaldehyde-fixed platelets was normal, and multimeric vWF analysis revealed a normal structure of patients' plasmatic vWF whatever the clinical stage in which it was studied. These findings suggest that, in spite of the existence of common clinical and biological features in the various forms of HUS, the pathogenesis of MMC-related HUS may be, at least in part, different from that of the other forms of HUS in which both platelet-aggregating activity and alterations in the vWF are found.

Two cases of von Willebrand disease (VWD) that revelaed an increased ristocetin-induced platelet aggregation (RIPA) and a qualitative abnormality of the factor VIII protein are reported. The threshold concentration of ristocetin giving a 30% increase in light transmission was 0.5 mg/ml in the proband and 0.4 mg/ml in her father (normal: 1.16 +/- SD 0.18 mg/ml) although both patients showed reduced plasma von Willebrand factor activity (VIIIR:WF). In both patients, the amount of factor VIII related antigen (VIIIR:AG) in their platelets were normal, but an increased binding affinity of platelets to plasma factor VIII was demonstrated. The qualitative abnormality of the factor VIII protein was characterized by an increasead anodal migration of VIIIR:AG in crossed immunoelectrophoresis (CIE), a delayed elution pattern as demonstrated by gel filtration on Sepharose 2B, and a decreased precipitation with concanavalin A (Con A). The response to DDAVP was also investigated.

The clinical picture, family history and laboratory findings of 100 patients with von Willebrand's disease (VWD) have been studied in Italy in a multicentre survey from two rural areas with a known high incidence of the disease and from two large cities (Rome and Milan). Bleeding time, procoagulant factor-VIII (VIIIAHF) assay, platelet retention and PRP-ristocetin aggregation were measured in each centre, and plasma samples were frozen and subsequently assayed for factor VIII-related antigen (VIIIAGN) and ristocetin co-factor (VIIIVWF) in one laboratory (Milan). On the basis of the inheritance pattern, clinical severity of the disease and laboratory findings, patients with VWD were separated into two groups. In 17 patients the absence of a family history of bleeding, a high incidence of parental consanguinity and involvement of both sexes suggested an autosomal recessive mode of inheritance; the unusual severity of the disease with markedly abnormal results of all six laboratory measurements (and frequent reduced levels of VIIIAGN and VIIIVWF in the unaffected parents) were consistent with the homozygous state. In 83 patients, the disease was familial (being transmitted as an autosomal dominant trait) and of moderate clinical severity. In these, VIIIVWF was usually much lower than VIIIAHF and VIIIAGN. The findings suggest that although some patients may present with simultaneous abnormalities of all six studied laboratory measurements, the majority show a variation in the spectrum of the abnormal results, without any obvious simple pattern.

The components of the factor VIII complex were estimated by immuno- and bioassays in 85 patients with liver disease. The plasma concentrations of the antigens were elevated in 65% (VIII:CAg) and in 76% (VIIIR:Ag) of patients while the biological activities were elevated in only 14% (VIII:C) and 15% (VIII:RiCof). There was no correlation with C-reactive protein, used as a measure of an acute phase reaction (X2 = 0.7; P = 0.1); or with severity of liver disease as judged by prothrombin ratio (P = 1.0) but highest values were observed in patients with cholestatic liver disease. Following parenteral vitamin K there was a significant fall in both the biological activity of VIIIC (36%) and of VIII:CAg (38%) in 13 vitamin K deficient patients (P less than 0.001) but no change in 23 vitamin K replete patients or in the VIIIR:Ag levels in either group. Factor V levels were lower in patients with parenchymal liver disease (0.54 +/- 0.1 units/ml, mean +/- SEM, n = 12; normal range 0.5-1.5 units/ml) than in patients with extrahepatic cholestasis who were vitamin K deficient (1.2 +/- 0.1 units/ml, P less than 0.0001). The levels of protein C antigen, the vitamin K dependent protease which inactivates factors VIII:C and V, was at the lower end of the range in both groups (0.7 +/- 0.1, mean +/- SEM, n = 18, normal range 0.74-1.4 units/ml). There was no significant change in either protein C antigen or factor V following vitamin K. The discrepancy between the biological activity of factor VIII and the antigen levels could represent accumulation of partially degraded factor VIII or production of a hypoactive form. There is no evidence that the reduction in VIIIC and VIII:CAg following vitamin K was mediated by protein C.

We have investigated platelet morphology and function in human maternal-newborn pairs. Fibrinogen concentration and factor-VIII activity in plasma were also determined. Our results showed that, compared to maternal platelets, neonatal platelets were poorly responsive to adenosine diphosphate, adrenaline and collagen. Uptake of labelled serotonin by neonatal and maternal platelets was the same, but release of the radioactivity was reduced in the former. Phagocytic activity of neonatal platelets, demonstrated with latex particles, was similar to that of maternal platelets. Although the ultrastructure of neonatal platelets approximated that of maternal platelets, immature appearing platelets were occasionally found in the neonatal samples. An unanticipated finding was that platelet aggregation induced by ristocetin was more vigorous in neonatal than in maternal platelet-rich plasma samples. Furthermore, neonal -lasma, which had lower fibrinogen and factor-VIII content than maternal plasma, facilitated maternal platelet aggregation by ristocetin and showed a greater ability than maternal plasma to promote ristocetin-induced aggregation of platelets of a patient with von Willebrand's disease. These results indicate that the plasma of neonates contains large quantities of the ristocetin-dependent platelet aggregation factor (RAF), probably more than is in maternal plasma, despite the higher levels of factor-VIII procoagulant activity in the latter. Thus, in the newborn, there is a clear dissociation between factor-VIII clotting activity and the RAF activity.

Antibodies against factor VIII collected from six patients were studied for their effect on factor-VIII coagulant activity, Willebrand factor activity (WF) and factor-VIII-related antigen. The results demonstrate differences between individual antibodies; some acting primarily as anti factor VIII, others inhibiting both after VIII and WF. Differences in effect between plasma and serum indicate that the process of coagulation alters the interaction of these antibodies.

To demonstrate the importance of the carbohydrate portions of bovine factor VIII and ristocetin for the induction of platelet aggregation, guinea pig platelets were treated with either borate or glycerol before and after addition of ristocetin or bovine factor VIII. Both borate and glycerol were capable of inhibiting the aggregation of blood platelets induced by adding bovine factor VIII or ristocetin to platelet-rich plasma. Furthermore, aggregates formed after treatment with either agent could be dispersed by high concentrations of either borate or glycerol. These effects did not appear to be solely the result of high ionic strength or of irreversible damage to the platelets. The results obtained support the hypothesis that the binding of the carbohydrate groups of factor VIII plays an important role in the induction of platelet aggregation and in the maintenance of aggregates formed in response to the treatment of platelet-rich plasma with bovine factor VIII or ristocetin.

The case of a patient with thrombocytopenia and giant platelets is presented. Platelet function tests including bleeding time, aggregation, availability of PF3, and serotonin uptake and release were abnormal. A bone marrow aspirate showed megakaryocytes that while not increased in number, were young in appearance. Electron microscopy of platelets revealed a dilated surface-connecting canalicular system and a prominent membrane complex. No similar findings were encountered among the patient's relatives. The various diagnostic possibilities in this patient are discussed.

Hybridoma antibodies specific for seven independent topographical sites were used to characterize von Willebrand factor (vWF) and to relate the epitopes to functional loci required for vWF-mediated adhesion of platelets to subendothelium and ristocetin-induced platelet aggregation. The capacity of antibodies to influence the adhesion of human platelets to rabbit aortic subendothelium was analysed in annular perfusion chambers. At a high shear rate similar to that of the microcirculation, four monoclonal antibodies inhibited adhesion. In contrast, no inhibition was observed at low shear. Only one of the four antibodies that inhibited platelet adhesion also attenuated ristocetin-cofactor activity (VIIIR:RCo). Conversely, one antibody that inhibited VIIIR:RCo had no effect upon platelet adhesion. These data support the hypothesis that the molecular loci involved in the two biological functions of vWF are not identical. When these conclusions are considered within the context of a spatial map of the vWF protein surface developed by competitive displacement analysis, the epitopes related to platelet adhesion appear to be spaced and differ from those involved in ristocetin-induced platelet-platelet interaction.

A case of acquired von Willebrand's syndrome (vWs) is described which appeared to be due to antibodies directed against factor VIII clotting activity (FVIIIC), factor VIII-related antigen (FVIIIRAg) and von Willebrand factor. The antibodies directed against FVIIIRAg was demonstrated by the inhibitory effect of a platelet eluate on Ristocetin-induced aggregation of normal platelets. This effect was not shown by the patient's platelet-poor plasma alone, nor could it be demonstrated in platelet eluates from 13 other patients who had antibodies to FVIIIC but in whom there was no evidence of an acquired vWs.

Five patients with an original diagnosis of von Willebrand's disease are described because of their levels of factor VIII related protein, Ristocetin-induced platelet aggregation and/or family studies differed from the main group of patients with classical von Willebrand's disease. Two had normal levels of factor VIII related protein with reduced Ristocetin aggregation when this was tested in platelet rich plasma. In one, however, this was due to a plasma defect and in the other to a platelet abnormality. After cryoprecipitate infusion all abnormal tests were corrected in both these patients. The first patient, however, failed to show a secondary rise of factor VIII whereas the second showed a secondary rise of both factor VIII and of factor VIII related protein. The other three cases, who were all very severely affected, have been separated from the main group as none of their families was segregating for classical von Willebrand's disease. It is suggested that the term von Willebrand's disease should be confined to those patients who have reduced factor VIII related protein and Ristocetin aggregation, and that von Willebrand's syndrome should be used for the various sub-groups that are emerging.

It has been reported that botrocetin, a Bothrops venom factor, induces platelet aggregation dependent on von Willebrand factor (vWF), and that platelet aggregation induced by Polybrene, a synthetic polycation, is enhanced by vWF. This report describes the platelet aggregability on stimulation with botrocetin and Polybrene in four patients with platelet-type von Willebrand disease (vWD) who showed increased platelet aggregation with low concentrations of ristocetin as the result of a platelet abnormality. Enhanced platelet aggregability with botrocetin was observed in platelet-rich plasma (PRP) from the patients. Platelet aggregation induced by botrocetin in a mixture of normal washed platelets and patient plasma was either decreased or normal, being dependent on the amount of plasma vWF. In contrast with ristocetin and botrocetin, Polybrene did not cause increased aggregation of patient PRP. Polybrene aggregated normal washed platelets less extensively in the presence of patient plasma than normal plasma. These studies demonstrated that botrocetin induced heightened interaction between platelets and vWF, but Polybrene did not, in platelet-type vWD, and that the enhanced responsiveness of patient platelets to botrocetin is related to an intrinsic platelet abnormality.

A variant of von Willebrand disease (vWD) has been identified in a 19-year-old woman with a severe bleeding syndrome. She had a very prolonged bleeding time (over 20 min), 24 U/dl factor VIII coagulant activity (F.VIII:C), 16 U/dl von Willebrand factor antigen (vWF:Ag), no ristocetin cofactor activity, and an anodal mobility of vWF:Ag on crossed immunoelectrophoresis (CIE). vWF:Ag was markedly reduced in her platelet lysate. In plasma and platelets, SDS-agarose electrophoresis consistently demonstrated the absence of large multimers, a relatively increased concentration of the fastest-moving multimer, and gross abnormalities of the internal structure of each vWF multimeric unit. Five members from the maternal side of the family had a double vWF:Ag peak by CIE and a relative increase of the fastest-moving vWF multimer by SDS-agarose electrophoresis; no quantitative or qualitative vWF defects were found in the paternal side of the family. The pattern of the findings in the propositus and her family is similar to those of type IIC vWD. However, there are some unique characteristics suggesting phenotypic variability in this subtype, such as low level of platelet vWF:Ag and the absence of increase of vWF after DDAVP administration.

We have studied four patients suffering from acquired von Willebrand's disease. All patients had a severe bleeding diathesis with recurrent life-threatening haemorrhages. Three of the patients had a monoclonal gammopathy and one of these developed multiple myeloma. In three patients tested, a plasma inhibitor to ristocetin cofactor activity was detected. In each case this was localized to the IgG fraction of plasma. In addition, VIII:C activity was found to be associated with the IgG fraction of patients' plasma and altered mobility of VIII:C was detected on Laurell immunoelectrophoresis. Furthermore, plasma from all four patients and the IgG fraction therefrom resulted in a dissociation of normal VIII:C into two components separable by gel-filtration on Sepharose 6B. Finally the circulating half-life of the three factor VIII activities was found to be markedly reduced in the patients with acquired von Willebrand's disease. We conclude that in the patients studied the coagulation defect was related to the presence of a circulating inhibitor to the factor VIII complex and that this inhibitor was associated with the IgG fraction of plasma.

A murine monoclonal antibody has been produced (RFF-VIII:R/2) that binds specifically to human factor VIII-related antigen (VIII:RAg) in plasma and in vascular endothelial cells but has no reactivity with factor VIII procoagulant antigen (VIII:cAg). This antibody is a potent inhibitor of von Willebrand factor activity (VIII:vWF) in that it can totally neutralize ristocetin-induced aggregation of platelet rich plasma and inhibit platelet adhesion at high flow rates. RFF-VIII:R/2 can be used in a one-stage, fluid phase immunoradiometric assay that can detect VIII:RAg at concentrations of 0.001 u/ml. This method has been used to analyse plasma from patients with von Willebrand's disease (vWD). Results obtained in these patients showed a high degree of correlation between the monoclonally-defined epitope and VIII:vWF levels measured by ristocetin-induced aggregation of washed platelets. This correlation was maintained in those patients with the 'variant' types of vWD who exhibit highly disparate VIII:vWF and VIII:RAg levels when the latter is determined using polyclonal antisera. It appears that this monoclonal antibody recognizes a site on the VIII:RAg molecule which is associated with its interaction with the platelet membrane. Immunoradiometric assays using RFF-VIII:R/2 offer a simplified, reproducible means of detecting functionally-active VIII:RAg as an alternative or supplement to techniques involving platelet interactions.

The effect of ristocetin on the binding of [125I]factor VIII to platelets was studied. High and low affinity F.VIII binding sites exist on platelets. The high affinity sites bind 13 times more F.VIII than the low affinity sites. Ristocetin increased the binding of F.VIII to both types of binding sites by increasing the affinity of F.VIII for the platelet and increasing the total number of platelet binding sites. Chymotrypsin-treated platelets were not aggregated by ristocetin and F.VIII: these platelets have less of the major platelet membrane glycoproteins and bind much less [125I]F.VIII than do buffer-treated platelets with and without ristocetin.