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Platelet adhesion to fibrillar collagens (types I, II, III, and V) and nonfibrillar collagens (types IV, VI, VII, and VIII) was investigated in the presence of physiologic concentrations of divalent cations under conditions of stasis and flow. Under static conditions, platelet adhesion was observed to collagen types I through VII but not to type VIII. Under flow conditions, platelet adhesion to collagen types I, II, III, and IV was almost independent of shear rates above 300/s. Collagen type V was nonadhesive. Platelet adhesion to collagen type VI was shear rate-dependent and optimal at a rate of 300/s. Collagen types VII and VIII showed minor reactivity and supported platelet adhesion only between shear rates 100 to 1,000/s. Monoclonal antibody (MoAb) 176D7, directed against platelet membrane glycoprotein Ia (GPIa; very late antigen [VLA]-alpha 2 subunit), completely inhibited platelet adhesion to all collagens tested, under conditions of both stasis and flow. Platelet adhesion to collagen type III at shear rate 1,600/s was only inhibited for 85%. The concentration of antibody required for complete inhibition of platelet adhesion was dependent on the shear rate and the reactivity of the collagen. An MoAb directed against GPIIa (VLA-beta subunit) partially inhibited platelet adhesion to collagen. These results show that GPIa-IIa is a major and universal platelet receptor for eight unique types of collagen.

The aim of this investigation was to identify domains of collagen type I that can support platelet adhesion under flow conditions. Four cyanogen bromide (CB) fragments composing 87% of the collagen alpha 1(I)-chain were studied under static and flow conditions. Under static conditions, bovine and human collagen fragment alpha 1(I)CB3 induced aggregate formation, whereas alpha 1(I)CB7 and alpha 1(I)CB8 supported adhesion of dendritic and contact platelets. Bovine alpha 1(I)CB6 weakly supported platelet adhesion. At shear rate 300/s, collagen fragment alpha 1(I)CB3 strongly supported platelet adhesion, whereas lower platelet adhesion was observed to alpha 1(I)CB7 and alpha 1(I)CB8. The fragment alpha 1(I)CB6 did not support platelet adhesion under flow conditions. Adhesion to alpha 1(I)CB3 was completely inhibited by a low concentration (0.6 IgG microgram/mL) of anti-GPIa monoclonal antibody (MoAb), whereas this concentration of antibody partially inhibited adhesion to alpha 1(I)CB7 and alpha 1(I)CB8. At higher concentrations (3 micrograms/mL) the anti-glycoprotein Ia (GPIa) antibody completely inhibited adhesion to alpha 1(I)CB8 and further reduced adhesion to alpha 1(I)CB7. Platelet adhesion to alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 was strongly inhibited by an anti-GPIb MoAb. A MoAb against the GPIb-binding site of von Willebrand factor (vWF) strongly inhibited platelet adhesion to alpha 1(I)CB7 and alpha 1(I)CB8, whereas platelet adhesion to alpha 1(I)CB3 was not inhibited. We conclude that under flow conditions alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 support GPIa/IIa-dependent platelet adhesion. The GPIb-vWF interaction is important under flow conditions for adhesion to alpha 1(I)CB7 and alpha 1(I)CB8 and probably also to alpha 1(I)CB3.

The aim of this investigation was to identify domains of collagen type I that can support platelet adhesion under flow conditions. Four cyanogen bromide (CB) fragments composing 87% of the collagen alpha 1(I)-chain were studied under static and flow conditions. Under static conditions, bovine and human collagen fragment alpha 1(I)CB3 induced aggregate formation, whereas alpha 1(I)CB7 and alpha 1(I)CB8 supported adhesion of dendritic and contact platelets. Bovine alpha 1(I)CB6 weakly supported platelet adhesion. At shear rate 300/s, collagen fragment alpha 1(I)CB3 strongly supported platelet adhesion, whereas lower platelet adhesion was observed to alpha 1(I)CB7 and alpha 1(I)CB8. The fragment alpha 1(I)CB6 did not support platelet adhesion under flow conditions. Adhesion to alpha 1(I)CB3 was completely inhibited by a low concentration (0.6 IgG microgram/mL) of anti-GPIa monoclonal antibody (MoAb), whereas this concentration of antibody partially inhibited adhesion to alpha 1(I)CB7 and alpha 1(I)CB8. At higher concentrations (3 micrograms/mL) the anti-glycoprotein Ia (GPIa) antibody completely inhibited adhesion to alpha 1(I)CB8 and further reduced adhesion to alpha 1(I)CB7. Platelet adhesion to alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 was strongly inhibited by an anti-GPIb MoAb. A MoAb against the GPIb-binding site of von Willebrand factor (vWF) strongly inhibited platelet adhesion to alpha 1(I)CB7 and alpha 1(I)CB8, whereas platelet adhesion to alpha 1(I)CB3 was not inhibited. We conclude that under flow conditions alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 support GPIa/IIa-dependent platelet adhesion. The GPIb-vWF interaction is important under flow conditions for adhesion to alpha 1(I)CB7 and alpha 1(I)CB8 and probably also to alpha 1(I)CB3.

Endothelial cells (EC) synthesize and secrete von Willebrand factor (vWF), a multimeric glycoprotein required for normal hemostasis. Within human endothelial cells, vWF multimers of extremely high molecular weight are stored in rod-shaped organelles known as Weibel-Palade bodies. Inflammatory mediators, such as interleukin-1, induce in vitro a variety of procoagulant responses by EC, including the secretion of stored vWF. We postulated that other inflammatory mediators might act to balance this procoagulant reaction, thereby assisting in the maintenance of blood fluidity during immune activation. Both gamma- interferon (gamma-IFN) and tumor necrosis factor (TNF) were found to act independently and cooperatively to depress the stimulated release of vWF from EC. Analysis of stored vWF in either gamma-IFN and/or TNF- treated EC demonstrated a loss of high molecular weight multimers while immunofluorescent studies documented a loss of visible Weibel-Palade bodies. This suggests that gamma-IFN and TNF interfere with normal vWF storage. gamma-IFN acted in a dose-, time-, and RNA-dependent fashion, and its inhibition of vWF release was reversible with time. No effect of gamma-IFN on EC was noted when anti-serum to gamma-IFN was added. Unlike gamma-IFN, alpha-interferon did not effect EC vWF. Therefore, gamma-IFN and TNF may be important in decreasing vWF release during inflammatory or immunologic episodes.

The Hermansky-Pudlak Syndrome (HPS) is an autosomal recessive inherited disorder characterized by oculocutaneous albinism, tissue accumulation of ceroid pigment, and a mild to moderate bleeding diathesis attributed to storage-pool deficient (SPD) platlets. Patients have platelet aggregation and release abnormalities. In addition, low levels of plasma von Willebrand factor (vWF) antigen in some HPS patients have been associated with a greater bleeding tendency than would be predicted from either condition alone. Other HPS patients have severe bleeding despite normal levels of plasma vWF, suggesting that at least one additional factor is responsible for their bleeding diathesis. Because platelet vWF levels have been well correlated with clinical bleeding times in patients with von Willebrand's disease, we have measured the platelet vWF activity and antigen levels in 30 HPS patients and have attempted to correlate their clinical bleeding with these values. The platelet vWF activity levels in patients was significantly lower than that of normal subjects (P0.0001). The patients as a group also had slightly lower values of plasma vWF activity when compared with normals (P-0.03). In 11 of the HPS patients, the multimeric structure of plasma vWF showed a decrease in the high molecular weight multimers and an increase in the low molecular weight multimers. In correlating the platelet and plasma vWF values with the bleeding histories, we were not able to show a predictable relationship in the majority of the patients.

Regional limb perfusion with antineoplastic agents stresses the local vasculature in a variety of ways. However, by monitoring the perfusates from limbs treated with melphalan alone or with melphalan plus tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma), we were able to distinguish the effect of the cytokines on the observed coagulant and fibrinolytic responses. We collected samples of effluent from a series of lower extremities that were perfused with the cytokines and/or melphalan as treatment for localized melanoma. Both regimens produced statistically significant evidence of coagulant and fibrinolytic activation. However, limbs receiving cytokines in addition to the melphalan responded with a sharper rise in tissue plasminogen activator (tPA) and plasmin (plasmin-antiplasmin complexes [PAP]) than limbs treated with melphalan alone. Evidence of thrombin formation (prothrombin fragment 1 + 2 [F1 + 2], thrombin-antithrombin complexes [TAT]) was also greater when the cytokines were included, although the response was delayed and less consistent than the fibrinolytic activation.

DDAVP is effective treatment in most types of von Willebrand's disease; however, in type 2B von Willebrand's disease the use of DDAVP has been contraindicated due to DDAVP-induced thrombocytopenia. Several reports have confirmed the thrombocytopenic effects of DDAVP and the presence of circulating platelet aggregates in type 2B von Willebrand's disease. We have infused three type 2B patients with DDAVP. The three patients had different mutations of their vWf. All three patients had a missense mutation which resulted in a single amino acid substitution in the disulfide loop of the A1 domain. Administration of 20 micrograms of DDAVP resulted in significant elevations of factor VIII, vWf antigen, and ristocetin cofactor levels. In contrast to other studies, DDAVP did not induce or enhance thrombocytopenia in these three patients. When blood was obtained by fingerstick and diluted into sodium oxalate (Unopette) or EDTA (Microvette), the platelet counts did not change over 4 hr. In contrast, blood collected directly into evacuated tubes containing sodium citrate, lithium heparin, or EDTA consistently demonstrated varying degrees of thrombocytopenia and platelet clumping. We also observed a shortening of the pre-infusion bleeding time over the 4 hr period. All three patients have been studied twice and each has shown consistent results. DDAVP appears to be a useful form of treatment in type 2B vWd.

Von Willebrand factor (vWf), a multimeric adhesive glycoprotein synthesized, stored, and secreted in megakaryocytes and endothelial cells, is normally found in plasma, platelets and subendothelium. While many substances mediate the release of vWf from endothelial cells, factors that enhance vWf synthesis and partitioning to its regulated pathway are currently unknown. We studied the effect of pharmacologic doses of heparin on the vWf content of endothelial cells. After a lag of 8 h and in the presence of crude or purified growth factor, heparin at doses between 0.25 and 2 U (1.4-11 micrograms)/ml, increased the content of high molecular weight vWf. The increased amounts of vWf were localized to Weibel-Palade bodies and extracellular matrix. Lower molecular weight highly sulfated heparin or heparin-like compounds were most active in growth factor dependent endothelial cell vWf expression. There was no clear importance of polysaccharide sequence or protein core.

Central venous access devices (VADs) are often associated with thrombotic obstruction of the axillary-subclavian venous system. To explore the accuracy of impedance plethysmography (IPG) in identifying this complication we performed IPG on 35 adult cancer patients before their VADs were placed and approximately 6 weeks later. At the time of the second IPG the patients also underwent contrast venography of the axillary-subclavian system. The venograms revealed partial venous obstruction in 12 patients (34%) and complete obstruction in two (5.7%). Although the IPG results from venographically normal and abnormal patients overlapped extensively, mean measurements of venous outflow were significantly lower in the patient population with abnormal venograms (P = 0.052 for Vo; P = 0.0036 for Vo/Vc). In our hands, therefore, upper extremity IPG cannot be used to make clinical decisions about individual patients with VADs, but it can distinguish venographically normal and abnormal populations.

T cells interact with the extracellular matrix via integrin receptors and these interactions affect both cellular localization and proliferation. The importance of these interactions in retrovirus-induced diseases, however, remains less clear. In the present study, we investigated changes in T cell adhesion to extracellular matrix proteins by HTLV-I expressing cell lines and human peripheral blood lymphocytes infected with HTLV-I by cocultivation. Cell lines and acutely infected primary peripheral blood lymphocytes demonstrated enhanced adhesion to fibronectin. Acute infection of peripheral blood lymphocytes increased the expression of alpha 5 beta 1 and alpha 4 beta 1 integrins. Antibodies to the alpha 4, alpha 5, and beta 1 subunits inhibited attachment of infected cells to fibronectin. We conclude that HTLV-I infection is associated with an increase in the expression of both the classical fibronectin receptor and the receptor for the alternatively spliced domain of fibronectin on peripheral blood lymphocytes. HTLV-I-related alterations in cell surface adhesion molecules may contribute to the abnormal proliferation of T cells in adult T cell leukemia (ATL) or to the abnormal localization of activated or infected T cells to the central nervous system of patients with tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM).

The antitrypanosomal drug suramin, which has recently been under investigation as a cancer chemotherapeutic agent, has previously been found to induce heparin-like anticoagulants in treated patients. In the currently reported work suramin is shown to have an additional anticoagulant activity that is due to direct effects of the drug on procoagulant proteins. The studies were conducted with pooled normal plasma treated in vitro with suramin and with plasma samples obtained from patients who had received the drug intravenously for 2 weeks. It is demonstrated that in plasma suramin inhibits factors V, VIII, IX, X, XI, and XII, while thrombin, prothrombin, and factor VII are unaffected. The inhibition of factor V is virtually irreversible, although the effect of suramin on the other factors is readily reversed by dilution.

We have studied a young male with lactoferrin deficiency and a bleeding tendency responsive to cryoprecipitate. This child has had increased bleeding following surgical procedures and a variably prolonged template bleeding time. The patient has a normal platelet count, normal in vitro platelet ATP secretion and aggregation in response to a variety of agonists, and normal concentration of plasma-von Willebrand factor ristocetin cofactor activity and antigen. Analysis of plasma-vWf multimers by agarose gel electrophoresis consistently demonstrated a subtle decrease in the largest vWf multimers. In contrast, analysis of the patient's platelet-vWf revealed normal vWf:Ag, decreased vWf ristocetin cofactor activity, and a striking absence of the high and intermediate size molecular weight vWf multimers. Analysis of surface bound platelet-vWf demonstrated normal amounts on the surface of unstimulated platelets, but after thrombin stimulation the platelet-vWf surface expression did not increase. This lack of increased platelet-vWf surface expression resulted from decreased binding of secreted platelet-vWf to be surface of stimulated platelets. These data suggest that the patient's bleeding tendency may be related to a defect in his platelet-vWf structure and/or mobilization. This case represents a unique demonstration of an abnormality of platelet-vWf in the presence of normal plasma-vWf, and supports the data indicating an important role for platelet-vWf in primary hemostasis.

Intracellular platelet fibrinogen surface expression was studied in arabinogalactan-purified, resting, and thrombin-stimulated platelets. Platelet fibrinogen is derived from endocytosis of plasma fibrinogen by megakaryocytes. Like a variety of other adhesive proteins, it is stored in the platelet alpha-granule. Platelet fibrinogen surface expression was studied by using the antigen-binding fragments of a murine monoclonal antibody to platelet fibrinogen, F26, and an immunopurified polyclonal antifibrinogen antibody. Studies correlating platelet fibrinogen surface expression with the presence of the glycoprotein IIb-IIIa (GPIIb-IIIa) complex showed that in the presence of ethylene glycol tetraacetic acid (EGTA) at 37 degrees C, neither the GPIIb-IIIa complex nor platelet fibrinogen was expressed on the surface of thrombin-activated platelets. Similar experiments performed in the presence of EGTA and calcium showed proportional expression of the GPIIb-IIIa complex and platelet fibrinogen. The addition of Arg-Gly-Asp-Ser-containing peptides, the pentadecapeptide of the fibrinogen gamma-chain carboxy terminus, or the monoclonal antibody 10E5, when directed against the GPIIb-IIIa complex before thrombin activation, inhibited 65% to 94% of the platelet fibrinogen expression, as determined with the polyclonal and monoclonal antigen-binding fragments. When these same inhibitory agents were added immediately after or 5 minutes after thrombin, the amount of inhibition decreased significantly. Similar studies with a washed platelet system revealed that when the inhibitors of platelet fibrinogen expression were added before thrombin stimulation, the degree of inhibition observed was only 24% to 38%. This suggests that the major portion of platelet fibrinogen expression involves the release of platelet fibrinogen and its subsequent binding to GPIIb-IIIa. This binding may occur within the open canalicular system or on the platelet surface; in either case, wherever the site of released platelet fibrinogen binding occurs, it can be markedly inhibited by the RGD-containing peptides and the gamma-chain fibrinogen peptides. Approximately 10% to 30% of platelet fibrinogen may be expressed prebound to a platelet receptor, or else it is released and binds to a platelet receptor other than the GPIIb-IIIa complex.

We used monoclonal antibodies against specific integrin subunits to examine the role of integrin receptors in keratinocyte activation. We found that before activation, beta 1 subunits in keratinocytes showed a diffuse distribution, whereas after activation, keratinocytes organized beta 1 receptors into marginal adhesion plaques. In immunoprecipitation experiments with antibodies against beta 1 integrin subunits, we found mostly immature subunits synthesized in keratinocytes freshly harvested from skin. Moreover, integrin receptor complexes immunoprecipitated from these cells by monoclonal antibodies against alpha 2, alpha 3, or alpha 5 subunits contained only immature beta 1 subunits. With keratinocytes cultured 4-7 days, anti-beta 1 antibodies immunoprecipitated mostly mature beta 1 subunits, and integrin complexes immunoprecipitated from cultured cells by anti-alpha subunit antibodies contained mostly mature beta 1 subunits. Antibodies directed against beta 1 subunits also inhibited keratinocyte migration. Based on these results, we suggest that up-regulation of migration by activated keratinocytes depends on changes in processing of pre-beta 1 subunits to mature beta 1 subunits. We also studied the distribution of integrin subunits in skin and on keratinocytes migrating out of skin explants. Whereas beta 1, alpha 2, and alpha 3 subunits were detected in keratinocytes in skin and migrating out of explants, alpha 5 subunits were observed only in migrating cells.

We describe a form of acute myeloid leukaemia (AML), designated AML-MO, with minimal myeloid differentiation, not included previously in the FAB classification. AML-MO cannot be diagnosed on morphological grounds alone as the blast cells are large and agranular, sometimes resembling L2 or, rarely, L1 lymphoblasts, and should be identified by the following features: negative myeloperoxidase (MPO) and Sudan Black B reaction (or positive in less than 3% of blasts), negative B and T lineage markers and expression of myeloid antigens recognized by at least one monoclonal antibody, CD13 or CD33. Other myeloid markers are also often positive and these include CD11b and the enzyme MPO demonstrated by immunocytochemistry and/or electron microscopy analysis. The findings in a group of 10 cases satisfying the criteria for AML-MO are described. AML-MO represents 2-3% of all cases of AML and 1-1.5% of all acute leukaemias. Its clinical and biological significance is not yet apparent but its identification in a larger number of cases may achieve this aim.

Unstimulated human platelets from normal volunteers adhere to sulfatides (galactosylceramide-I3-sulfate) as single cells but do not adhere appreciably to other lipids including gangliosides, neutral glycolipids, phospholipids or cholesterol-3-SO4. Platelet adhesion to sulfatide is saturable and dose-dependent, reaches maximal levels in 90 to 120 min, and is not divalent cation-dependent. Because sulfatides bind von Willebrand factor (vWf) with specificity and high affinity and platelet adhesion to structurally related sulfated glycolipids is approximately proportionate to their ability to bind vWf, we examined whether vWf mediates platelet adhesion to sulfatides. Platelets from a patient with severe Type I von Willebrand's disease adhere poorly to sulfatides. However, adhesion to levels seen with normal platelets is restored by the addition of vWf. Adhesion of normal platelets can be partially inhibited by a monospecific antibody to vWf. Normal platelet adhesion to sulfatides, however, is not increased following preincubation with vWf. Both vWf binding and platelet adhesion to sulfatides can be inhibited by the sulfated polysaccharide dextran sulfate at low concentration, fucoidan at high concentrations, but not by heparin, fibrinogen, fibronectin, or the synthetic peptides Gly-Arg-Gly-Asp-Ser-Pro or Gly-Arg-Gly-Glu-Ser-Pro. Thus, adhesion to sulfatides appears to be of two types; vWf dependent (50-75%) and vWf independent (25-50%).

Type IIB von Willebrand Disease (vWD) is characterized by the selective loss of large von Willebrand Factor (vWF) multimers from plasma, presumably due to their increased reactivity with platelets and subsequent clearance from the circulation. Using the PCR, one of a panel of four potential missense mutations was identified in each of the 14 patients studied from 11 unrelated families. None of these substitutions was encountered in a large panel of normal DNAs. These changes all represent C----T transitions at CpG dinucleotides, proposed "hot spots" for mutation in the human genome. The four resulting amino acid substitutions, Arg543----Trp, Arg545----Cys, Val553----Met, and Arg578----Gln, are all clustered within the GpIb binding domain of vWF. Disruption of this latter functional domain may explain the pathogenesis of Type IIB vWD. By sequence polymorphism analysis, the Arg543----Trp substitution was shown to have occurred as at least two independent mutational events. This latter observation, along with the identification of mutations in all 14 patients studied and their localization to the GpIb binding domain, all strongly suggest that these substitutions represent the authentic defects responsible for Type IIB vWD. This panel of mutations may provide a useful diagnostic tool for the majority of patients with Type IIB vWD.

The factor VIII/von Willebrand factor (f.VIII/vWf) protein was purified from the plasma of a patient with von Willebrand's disease (vWd). The patient had all of the classic laboratory findings of vWd except for the ristocetin-induced platelet aggregation of his own platelet-rich plasma. The disease has been documented in three generations. Comparison of the purified normal and vWd f.VIIi/vWf protein revealed several abnormalities, including decreased concentration of f.VIII/vWf antigen; decreased specific vWf activity; absence of the larger molecular forms of the f.VIII/vWf protein; carbohydrate deficiencies affecting the sialic acid, penultimate galactose and N- acetylglucosamine moieties; and decreased binding of the f.VIII/vWf protein to its platelet receptor. These studies indicate the multiplicity of biochemical and functional abnormalities associated with the f.VIII/vWf protein in vWd. f.VIII/vWf protein to normal f.VIII/vWf protein that had been treated with 2-mercaptoethanol (2-ME) to reduce the multimer size and then treated with specific exoglycosidases to remove the sialic acid and penultimate galactose residues revealed similar biologic properties.

A bleeding diathesis is described which is phenotypically indistinguishable from hemophilia A and which has been transmitted as a dominant trait in three generations of women in a North Carolina kindred. The abnormal phenotype is characterized by clinical mildness and slightly abnormal clotting time, prothrombin consumption, and partial thromboplastin time. Bleeding time, platelet count, clot retraction, tourniquet test, and prothrombin time are normal. Concentration of factors I, II, V, VII, IX, X, and XII are normal, while factor VIII activity is reduced to 2%-5% of control values. De novo synthesis of factor VIII does not occur after transfusion; factor VIII-related antigen is normal; patients'plasmas aggregate platelets normally in the presence of ristocetin, and a typical protein pattern is seen when a chymotryptic digest of cryoprecipitate of the proband is examined by SDS-polyacrylamide gel electrophoresis. Six possible genetic explanations are entertained. Balanced X-autosomal translocation of hemophilia A heterozygotes has been excluded by cytogenetic analysis of metaphase chromosomes. Classes von Willebrand's disease (vWd) is probably excluded on the basis of the laboratory data, and extreme lyonization of hemophilia A heterozygotes on probabilistic grounds. The genetic possibilities which cannot be excluded include a previously unrecognized variant mutation at the vWd locus, a dominant mutation at the hemophilia A locus on the X chromosome, and dominant mutation at a hypothetical fourth locus involved in factor VIII synthesis and control.

The adhesive glycoproteins laminin, thrombospondin, and von Willebrand factor bind specifically and with high affinity to sulfated glycolipids. These three glycoproteins differ, however, in their sensitivity to inhibition of binding by sulfated monosaccharides and polysaccharides. Heparin strongly inhibits binding of thrombospondin but only weakly inhibits binding of laminin and von Willebrand factor. Fucoidan strongly inhibits binding of both laminin and thrombospondin but not of von Willebrand factor. Laminin shows significant specificity for inhibition by monosaccharides, whereas thrombospondin does not. Thus, specific spacial orientations of sulfate esters may be primary determinants of binding for the three proteins. Laminin, thrombospondin, and von Willebrand factor also differ in their relative binding affinities for purified sulfated glycosphingolipids. The three proteins strongly prefer terminal-sulfated lipids and bind only weakly to sulfated gangliotriaosyl ceramide with a sulfate ester on the penultimate galactose. Thrombospondin binds with highest affinity to galactosyl sulfatide but only weakly to more complex sulfatides, whereas von Willebrand factor prefers galactosyl sulfatide but binds with moderate affinity to various sulfated glycolipids. Laminin also is less selective than thrombospondin but is less sensitive for detection of low sulfatide concentrations. Galactosyl sulfatide at 1-5 pmol can be detected by staining of lipids separated on high performance TLC with 125I-thrombospondin or 125I-von Willebrand factor. 125I-von Willebrand factor was examined as a reagent for detecting sulfated glycolipids in tissue extracts. Rat kidney lipids contain 5 characterized sulfated glycolipids: galactosyl ceramide I3-sulfate, lactosyl ceramide II3-sulfate, gangliotriaosyl ceramide II3-sulfate, and bis-sulfated gangliotriaosyl and gangliotetraosyl ceramides. von Willebrand factor detects all of these lipids as well as several additional minor sulfated lipids. Complex monosulfated lipids are detected in several human tissues including kidney, erythrocytes, and platelets by this technique.

Five patients with glycogen storage disease type I (GSD-I) were evaluated for a bleeding diathesis and subsequently were given an infusion of 1-deamino-8-D-arginine vasopressin (DDAVP). Although platelet counts were normal or slightly elevated, the baseline template bleeding times were prolonged in four of the patients. Prothrombin times and activated partial thromboplastin times were normal, while ADP- and epinephrine-induced platelet aggregations were absent in the three patients tested. Ristocetin- and collagen-induced platelet aggregations were abnormal. Laurell and immunoradiometric determinations of the factor VIII-related antigen (vWf antigen) were decreased. Glyoxyl agarose gel electrophoresis of the patients' plasma revealed abnormal multimer patterns in four of the five patients. After the DDAVP infusion the platelet aggregation abnormalities persisted; however, the bleeding time and the von Willebrand antigen and activity corrected. We conclude that GSD-Ia patients may have a metabolically acquired form of von Willebrand's syndrome as well as an acquired intrinsic platelet defect, and that DDAVP may be useful in the management of bleeding in these patients.

Synthetic peptides containing the adhesion site recognition sequences present on the A alpha and gamma chains of fibrinogen were studied for their effect on the binding of endogenous platelet-von Willebrand factor (vWF) and exogenous plasma-vWf to thrombin-stimulated platelets. In agreement with previously reported data, the tetrapeptide consisting of the RGDS sequence was a more potent inhibitor of plasma-vWf binding to platelets than was the pentadecapeptide of the carboxy terminus of the fibrinogen gamma-chain (IC50 10.6 mumol/L for the RGDS tetrapeptide v 44.9 mumol/L for the gamma-chain pentadecapeptide). No apparent synergy in the inhibition of plasma-vWf binding was noted when the RGDS and gamma-chain peptides were used together (IC50 15.2 mumol/L). In contrast, the gamma-chain peptide was significantly more inhibitory than was the RGDS tetrapeptide on the binding of platelet-vWf to platelets (IC50 1.4 mumol/L for the gamma-chain pentadecapeptide v 4.5 mumol/L for the RGDS tetrapeptide, P less than .05), and there was significant synergy in the inhibition of platelet-vWf binding noted when the gamma-chain and RGDS peptides were used together (IC50 0.04 mumol/L). These results indicate that the binding of platelet-vWf to its receptor on the platelet glycoprotein IIb/IIIa complex involves both the RGDS and gamma-chain recognition sites. In contrast to the results with plasma-vWf binding, the gamma-chain recognition site appears to be more important than the RGDS recognition site in platelet- vWf binding to platelets.

Two monoclonal antibodies--one that blocks ristocetin-induced platelet binding of von Willebrand factor to glycoprotein Ib and one that blocks adenosine diphosphate-induced binding of fibrinogen to the glycoprotein IIb/IIIa complex--were used to assess the binding site(s) for von Willebrand factor when platelets are stimulated with thrombin or adenosine diphosphate (ADP). Neither agonist induced binding of von Willebrand factor to glycoprotein Ib. ADP and thrombin induced von Willebrand factor binding exclusively to the glycoprotein IIb/IIIa complex. The results of the site of binding of von Willebrand factor with thrombasthenic platelets were consistent with the data obtained with the monoclonal antibodies and normal platelets. Human fibrinogen caused complete inhibition of thrombin-induced von Willebrand factor binding to normal platelets at concentrations considerably below that found in normal plasma. We conclude that thrombin induces very little binding of exogenous von Willebrand factor to platelets at normal plasma fibrinogen levels.

Studies of the prothrombin time (PT) have revealed that contact with borosilicate or commercial siliconized borosilicate markedly shortens the PT. This shortening is related to the activation of the contact phase of blood coagulation. This shortening of the PT occurs in both normal whole blood and plasma when stored in borosilicate or siliconized borosilicate tubes at 4 degree C and to a lesser degree at room temperature. Studies indicated the importance of several coagulation factors in decreasing the PT. The PT did not change in blood deficient in factor XII or in plasma deficient in Fletcher factor or high molecular weight kininogen, while blood deficient in CI esterase inhibitor (CI INH) had the most profound shortening. Shortening of the PT correlated directly with increased levels of factor VII. When purified CI INH was added to normal blood, it markedly reduced the activation of factor VII and the shortening of the PT in a dose-related manner. These studies indicate the pivotal roles of the contact phase of coagulation in initiating activation of the PT and of CI INH in inhibiting the activation of the coagulation factor(s) responsible for the cold-promoted activation of factor VII.

Quantitative ristocetin-induced platelet aggregation of normal platelet- rich plasma (PRP) decreased with time after PRP preparation. An increase in p H of the PRP with time proved to be responsible for this finding. Diffusion of CO2from the plasma is the prime determinant of the change in pH. Since a complex combination of factors influences CO2 diffusion (surface area-to-volume relationship, capping, mixing, etc.) The change in pH is variable with time. Thus, quantitative ristocetin aggregation should be pH controlled. A simple device for maintaining PRP pH constant by control of the ambient pCO2 was designed and found effective in keeping both pH and quantitative ristocetin aggregation constant over a prolonged period of time. It can be adapted for use in platelet aggregation studies employing other reagents. The pH dependence of ristocetin-induced platelet aggregation is consistent with other data supporting an elctrostatic interaction between the platelet, von Willebrand factor, and ristocetin. We favor a model wherein ristocetin neutralizes some of the platelet's negative change and permits the von Willebrand factor to bridge sites on separate platelets to induce agglutination.

The factor VIII/von Willebrand factor protein was radiolabeled after modification by galactose oxidase and reduction with tritiated potassium borohydride. This mild efficient method for labeling resulted in retention of over 90% of the biologic activities of the factor VIII/von Willebrand factor protein. Binding of this protein to platelets was found to be specific, and binding sites could be saturated in the presence of ristocetin. However, binding was highly dependent on ristocetin concentration, as the number of human factor VIII/von Willebrand factor molecules bound per platelet was a function of the ristocetin concentration. At a ristocetin concentration of 0.55 mg/ml, each platelet binds approximately 11,000 factor VIII/von Willebrand factor molecules per platelet. Scatchard analysis of the concentration-dependent binding sites yielded a hyperbolic plot that appeared to be related to the existence of two classes of binding sites. The higher affinity class had a Kd of 3.7 x 10(-10) M 3500 sites/platelet, while the lower affinity class had a Kd of 2.35 x 10(- 9) M and a capacity of 7500 sites/platelet. As with ristocetin-induced platelet agglutination, the carbohydrate content plays a significant role in the binding of the factor VIII/von Willebrand factor protein to the platelet.

SummaryA multicenter study of various types of von Willebrand’s disease (vWD) was conducted in order to compare the different electrophoretic techniques used to evaluate von Willebrand factor multimers in plasma. Seven laboratories participated in the blind study of eight plasma samples from two healthy subjects and six vWD types and subtypes (la, lb, IIA, IIB, IIC and IID). From the results of the multimeric analysis of these samples, it appears that the differential diagnosis of vWD types and subtypes should be first approached by using a low-resolution electrophoretic technique, where each vWF multimer appears as a single band. Low-resolution techniques differentiate type I from type II, subtype la from lb and also subtype IIA from other type II subtypes. When type II subtypes other than IIA are identified with these techniques, samples should be rerun using high resolution techniques that resolve each of the fastest migrating multimers in at least three subbands, and permit the differentiation of subtypes IIB, IIC and IID.

Plasma exchange has been proposed as a treatment for multiple disorders. Three patients with amyotropic lateral sclerosis, who were hemostatically normal, were studied through a total of 11 4-liter exchanges. Plasma was replaced by an equal volume of 5% albumin or 5% plasma protein fraction. Serial studies revealed that immediately after the exchange transfusion, there was significant prolongation of the prothrombin, partial thromboplastin, and thrombin times with reduction of the fibrinogen and antithrombin III levels. Factors V, VII-X, IX, and X were all significantly decreased, as were the factor VIII antigen, procoagulant, and the ristocetin cofactor activities. Platelet counts were obtained before and after exchanges and revealed significant decreases. Four hours after exchange, all parameters remained abnormal except the factor IX, ristocetin cofactor, and factor VIII procoagulant activities. By 24 hr, all hemostatic parameters had returned to normal. These studies indicate that plasma-exchange transfusion with material devoid of coagulation factors results in a coagulation defect that may be of clinical significance in a hemostatically compromised patient.

A bleeding diathesis is described which is phenotypically indistinguishable from hemophilia A and which has been transmitted as a dominant trait in three generations of women in a North Carolina kindred. The abnormal phenotype is characterized by clinical mildness and slightly abnormal clotting time, prothrombin consumption, and partial thromboplastin time. Bleeding time, platelet count, clot retraction, tourniquet test, and prothrombin time are normal. Concentration of factors I, II, V, VII, IX, X, and XII are normal, while factor VIII activity is reduced to 2%-5% of control values. De novo synthesis of factor VIII does not occur after transfusion; factor VIII-related antigen is normal; patients'plasmas aggregate platelets normally in the presence of ristocetin, and a typical protein pattern is seen when a chymotryptic digest of cryoprecipitate of the proband is examined by SDS-polyacrylamide gel electrophoresis. Six possible genetic explanations are entertained. Balanced X-autosomal translocation of hemophilia A heterozygotes has been excluded by cytogenetic analysis of metaphase chromosomes. Classes von Willebrand's disease (vWd) is probably excluded on the basis of the laboratory data, and extreme lyonization of hemophilia A heterozygotes on probabilistic grounds. The genetic possibilities which cannot be excluded include a previously unrecognized variant mutation at the vWd locus, a dominant mutation at the hemophilia A locus on the X chromosome, and dominant mutation at a hypothetical fourth locus involved in factor VIII synthesis and control.

The characteristics of the intact factor VIII/von Willebrand factor protein binding to human platelets was compared to 2-mercaptoethanol- treated factor VIII/von Willebrand factor protein and to fractions of plasma factor VIII/von Willebrand factor protein that elute after the void volume. These studies indicate that the factor VIII/von Willebrand factor protein larger size oligomers bind preferentially with high affinity to low capacity sites on human platelets. The intermediate and smaller size oligomers bind with intermediate or low affinity to sites with a much greater capacity. The results from binding analysis are also paralleled by the competitive inhibition of the intact factor VIII/von Willebrand factor protein by the various 2-mercaptoethanol- treated materials. These studies indicate that the two classes of binding sites seen in previous reports of factor VII/von Willebrand factor binding reflect heterogeneity in the oligomer size of the factor VIII/von Willebrand factor protein used in these assays. This study provides a model for understanding some of the normal structure- function relationships of the normal factor VIII/von Willebrand factor protein and the defect(s) in a variant form of von Willebrand's disease. In this form of the disease, decreased factor VIII/von Willebrand factor binding to platelets is reflected in decreased von Willebrand factor activity but coagulant and/or antigen levels are normal or only slightly decreased.

Peripheral blood, bone marrow films, and bone marrow biopsy specimens from 110 patients, well characterised by clinical and laboratory studies, including electron microscopy, were reviewed, to determine proposals for the classification of chronic (mature) B and T cell leukaemias. On the basis of cytology and membrane phenotype the following disorders were defined: (i) B cell type: chronic lymphocytic leukaemia (CLL); CLL of mixed cell type, which includes cases with more than 10% and less than 55% prolymphocytes (CLL/PL), and a less well defined form with pleomorphic lymphocytes but less than 10% prolymphocytes; prolymphocytic leukaemia (PLL); hairy cell leukaemia (HCL); HCL variant; splenic lymphoma with circulating villous lymphocytes; leukaemic phase of non-Hodgkin's lymphoma (follicular lymphoma, intermediate, or mantle zone lymphoma and others); lymphoplasmacytic lymphoma with peripheral blood disease (mostly Waldenström's macroglobulinaemia); and plasma cell leukaemia. (ii) T cell type: T/CLL, which was differentiated from reactive T/lymphocytosis; T/PLL; adult T cell leukaemia/lymphoma; and Sézary's syndrome. The recognition of distinct entities within the B and T cell leukaemias seems to have clinical and epidemiological connotations. It is hoped that these proposals may serve as the basis for further work, discussion, and improved management of patients.

A dysfibrinogenemia (fibrinogen Bethesda) was detected in a 9 yr old male of Mexican-English extraction who had a lifelong history of mild bleeding diathesis. The prothrombin and partial thromboplastin times were moderately prolonged; the thrombin and Reptilase times were markedly prolonged. The plasma fibrinogen level was normal by conventional methods but was markedly reduced by the Clauss method. Results of all other tests for clotting factors, fibrinolysis, antithrombin levels, clot stabilization, and fibrin(ogen) degradation products were normal. The patient's plasma and fibrinogen inhibited the clotting of normal plasma or fibrinogen by thrombin. Family studies revealed that the propositus' mother and two siblings exhibited these abnormalities to a lesser degree and indicated an autosomal dominant inheritance. Fibrinogen Bethesda was similar to normal fibrinogen in the following respects: metabolic turnover time (measured in the propositus' mother); immunodiffusion, ultracentrifugal, electrophoretic (on cellulose acetate or polyacrylamide gel), and chromatographic (on DEAE-cellulose) characteristics; sialic acid content; and aggregation of fibrin monomers. By contrast, fibrinogen Bethesda gave an abnormal immunoelectrophoretic pattern especially when whole plasma (as opposed to purified fibrinogen) was examined, and it showed a pronounced decrease in the rate of fibrinopeptide release by thrombin. This decrease, which was shown to involve both fibrinopeptides A and B, distinguishes fibrinogen Bethesda from previously reported dysfibrinogenemias.

A total of 337 measurements of urinary fibrin(ogen) split products (FDP) have been made over a 2-year period on 56 patients with lupus nephritis. Nineteen patients were studied during a 14 week controlled trial of cytoxic drug therapy and the remaining patients were studied at random intervals. An attempt was made to correlate FDP excretion levels with accepted parameters of renal disease and of lupus activity. While patients with significant nephritis by other criteria excreted elevated levels of FDP when compared with normal individuals or patients with rheumatoid arthritis, the excretion by individual patients fluctuated markedly in sequential measurements. In addition FDP excretion correlated poorly with BUN, creatinine clearance, and protein excretion, and with indicators of lupus activity (C3 and anti-DNA levels). We conclude that although fibrin deposition in the glomerulus is almost certainly an important factor in the pathogenesis of lupus nephritis, urinary FDP levels are not helpful in assessing the progress of glomerular damage in individual patients.

SummaryA dysfibrinogenemia (fibrinogen Bethesda II) was recognized in a Caucasian family with no history of bleeding or thrombosis. Thrombin and Reptilase times were prolonged, and there was a discrepancy between the fibrinogen levels determined by the fibrin tyrosine content and the Clauss method. Other coagulation and fibrinolytic measurements yielded normal values. The half-life of homologous and autologous 125I-fibrinogen in the propositus was approximately 20% shorter than the mean (3.70 ±0.45 days) of normal individuals. Fibrinogen Bethesda II exhibited normal migration during electrophoresis in polyacrylamide gel (in the presence of sodium dodecyl sulfate) or cellulose acetate, normal ultracentrifugal behavior, a normal chromatographic elution pattern and normal levels of hexose and sialic acid. Upon immunoelectrophoresis of whole plasma, the arc was more anodal than that produced by the fibrinogen of normal plasma. Functionally, fibrinogen Bethesda II prolonged the thrombin and Reptilase times of normal plasma and fibrinogen. The rate at which fibrinopeptides were released by thrombin or ancrod was similar to that for normal fibrinogen, and the amounts eventually reached the same levels, but a slight delay was consistently observed in the early stages of the reaction. Aggregation of fibrin monomers prepared with either thrombin or Reptilase was markedly abnormal, whereas the crosslinking (as assessed by polyacrylamide gel electrophoresis) did not differ from that of normal fibrin.

The adhesive glycoproteins laminin, thrombospondin, and von Willebrand factor bind specifically and with high affinity to sulfated glycolipids. These three glycoproteins differ, however, in their sensitivity to inhibition of binding by sulfated monosaccharides and polysaccharides. Heparin strongly inhibits binding of thrombospondin but only weakly inhibits binding of laminin and von Willebrand factor. Fucoidan strongly inhibits binding of both laminin and thrombospondin but not of von Willebrand factor. Laminin shows significant specificity for inhibition by monosaccharides, whereas thrombospondin does not. Thus, specific spacial orientations of sulfate esters may be primary determinants of binding for the three proteins. Laminin, thrombospondin, and von Willebrand factor also differ in their relative binding affinities for purified sulfated glycosphingolipids. The three proteins strongly prefer terminal-sulfated lipids and bind only weakly to sulfated gangliotriaosyl ceramide with a sulfate ester on the penultimate galactose. Thrombospondin binds with highest affinity to galactosyl sulfatide but only weakly to more complex sulfatides, whereas von Willebrand factor prefers galactosyl sulfatide but binds with moderate affinity to various sulfated glycolipids. Laminin also is less selective than thrombospondin but is less sensitive for detection of low sulfatide concentrations. Galactosyl sulfatide at 1-5 pmol can be detected by staining of lipids separated on high performance TLC with 125I-thrombospondin or 125I-von Willebrand factor. 125I-von Willebrand factor was examined as a reagent for detecting sulfated glycolipids in tissue extracts. Rat kidney lipids contain 5 characterized sulfated glycolipids: galactosyl ceramide I3-sulfate, lactosyl ceramide II3-sulfate, gangliotriaosyl ceramide II3-sulfate, and bis-sulfated gangliotriaosyl and gangliotetraosyl ceramides. von Willebrand factor detects all of these lipids as well as several additional minor sulfated lipids. Complex monosulfated lipids are detected in several human tissues including kidney, erythrocytes, and platelets by this technique.

The presenting clinical characteristics and the results of therapy in 30 cases of American Burkitt's lymphoma are described. Five patients presented with localized disease. The abdomen was the most frequent site of involvement (19 cases). Serum lactic dehydrogenase (LDH) levels closely correlated with extent of tumor mass. Of the 22 patients treated with large doses of parenteral cyclophosphamide, complete remission was achieved in 13 (59 per cent). Of these only four have had a relapse, all within 12 months of treatment. The remainder are alive, free of disease and have not received any treatment for up to 80 months or more. The site and volume of tumor mass predicted for prolonged survival. None of the six patients with bone marrow or central nervous system involvement remained tumor-free. A complete remission was achieved in 8 of 9 patients with presenting LDH levels of less than 700 IU/ml and they have remained free of disease, whereas only 4 of 13 patients with LDH levels greater than 700 IU/ml had a complete response and 3 of these had a relapse within 12 months. In six cases, the massive tumor regression following chemotherapy was associated with serious metabolid consequences including hyperkalemia (six cases), hypocalcemia, hyperphosphatemia (one case) and lactic acidosis (one case). There were four sudden deaths in less than 48 hours after chemotherapy; two of these were attributable to hyperkalemia. In all cases therw were large tumor masses and/or elevated serum LDH levels.

The antibiotic vancomycin shares many similarities with ristocetin, an agent noted for its effects on platelets and plasma fibrinogen. Vancomycin did not aggregate platelets as ristocetin, but platelets were incorporated into precipitates induced by vancomycin. Fibrinogen and factor VIII were precipitated from plasma at low concentrations of vancomycin. The precipitated fibrinogen remained clottable. Hepatitis B surface antigen was selectively precipitated from serum and could be recovered from the precipitate. Rabbits receiving bolus intravenous injections of high doses of vancomycin developed hypofibrinogenemia and thrombocytopenia within minutes and often went on to die. Studies with 125I-vancomycin revealed little stable binding of the antibiotic to platelets or fibrinogen. A relationship is suggested between the potent protein precipitating effects and phlebitis at the infusion site commonly associated with vancomycin therapy.

A patient with polyneuropathy due to Waldenstrom9s macroglobulinemia (WM) was treated successfully with chlorambucil and prednisone. Before therapy, 60% of peripheral lymphocytes were B cells, the nerve had IgMK-bearing B-cell infiltrates, and the circulating IgM had antibody-binding activity to autologous and homologous nerves. Neurologic improvement, sustained for 4 years, began 3 months after therapy and coincided with the return to normal of bone marrow and circulating B cells. Binding of IgM to autologous and homologous nerves persisted after therapy, suggesting that not the IgM alone but other B-cell factors, possibly complexed to IgM, may have been responsible for the nerve damage.

Functional properties of the carbohydrate chain of human thrombin were examined by quantitating the activity of the enzyme before and after partial removal of its oligosaccharide by exoglycosidases. The following activities were studied: fibrinogen clotting, factor VIII coagulant (VIII:C) activation, inhibition by defibrinated plasma (anti-thrombin-III and alpha 2-macroglobulin), binding to polymerized fibrin, and stimulation of platelet release and aggregation. In general, the published information about the activity of native thrombin in these interactions was confirmed, though differences were observed in the association constants describing thrombin binding to fibrin. Partial deglycosylation had no apparent effect on any of these activities. It is concluded, therefore, that the oligosaccharide of human thrombin is located outside the major protein and platelet-binding regions of the molecule.

1-D-Amino(8-D-arginine)-vasopressin (DDAVP) infusion in three patients with type IIa von Willebrand's disease (vWD) resulted in a normalization of the factor VIII coagulant, factor VIII-related antigen, and von Willebrand factor (vWF) (ristocetin cofactor) activities and the bleeding time. The normalization of these hemostatic parameters persisted for four hours. Over the same time period there was a marked increase in the quantity of the vWF multimers when blood was collected in the presence of protease inhibitors. The vWF multimers present were even larger than the normal. When blood was collected in the absence of protease inhibitors, a smaller increase in the plasma vWF multimers was observed and fewer of the intermediate and larger vWF multimers were seen; multimers larger than those present in normal plasma were not visualized. The platelet vWF multimers and activities did not change with or without inhibitors. These studies suggest that there is a subgroup of patients with type IIa vWD who respond to DDAVP with complete normalization of their hemostatic abnormalities and whose vWF is sensitive to proteolysis.

Seven cases of acute nonlymphocytic leukemia (ANLL) and one case of a malignant myeloproliferative syndrome have been seen after extensive radiation therapy for non-Hodgkin's lymphoma or chronic lymphocytic leukemia. A myeloproliferative syndrome with abnormalities in granulocytic, erythrocytic, and thrombocytic cell lines was present in all patients and in seven patients preceded ANLL by 2--18 months. The median time to the development of ANLL after primary disease therapy was 61 months (33--98 range). The leukemia was extremely refractory to therapy and median survival after diagnosis of ANLL was two months (range 0--9 months). Leukemia was seen only in those patients who received multiple courses and multiple techniques of radiation therapy.

Decreased quantitative RPA of PRP is used to distinguish patients with von Wille-brand’s disease from normals and hemophiliacs. We observed that RPA (1.8 mg/ml final concentration) of PRP from 9 normals varied markedly with time after PRP preparation. In 6/9 the initial RPA was greatest and decreased progressively with time. In 3/9 RPA increased over the first half hour and then decreased progressively. The decrease from peak values was 37 ±12% (mean±1 SD) at 1 hr, 69 ±20% at 2 hr and 70±10% at 2.5 hr.Several data suggest a role for pH in this phenomenon: (1) pH of the PRP’s increased with time and the decreases in RPA paralleled the increases in pH (2) maintaining the PRP under a 5% CO2-95% O2 atmosphere prevented the rise in pH and lessened the decrease in RPA over time (3) rapidly increasing the pH to 8.3 by bubbling nitrogen through the PRP or titrating with base decreased RPA (81% and 55%) (4) immediate back titration restored the RPA.We conclude that quantitative RPA changes with time after PRP preparation and this is probably in part a function of pH. Since pH change with time is variable for different PRP preparations, choosing an arbitrary time limit after PRP preparation for performing studies is an inadequate control. Quantitative RPA should be pH controlled.