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Studies of platelet C1 have been performed in 11 patients with chronic lymphocytic leukemia (CLL). The platelet C1 level evaluated by the hemolytic method was very low and the platelet aggregation induced by aggregated IgG or anti C1q antisera was significantly reduced. "In vitro", the lymphocytes from CLL were capable of inhibiting platelet C1 and platelet aggregation induced by aggregated IgG and anti C1q to a greater extent than normal lymphocytes. These results lead the authors to discuss the role of platelet C1 in the interaction platelet/immunoglobulins and the nature of the inhibition.

The effect on platelet function of a monoclonal platelet antibody to platelet membrane glycoprotein I was tested. This antibody, AN51, inhibited ristocetin or bovine factor VIII-induced aggregation but did not modify ADP, collagen type I or type III, thrombin or arachidonic acid induced aggregations. Furthermore, the adhesion-aggregation of platelets induced by microfibrils was also inhibited by the antibody. Platelet adhesion to rabbit aorta subendothelium was impaired by the antibody. The persistent adhesion of platelets to collagenase-treated subendothelium was also inhibited. These findings strongly suggested that platelet membrane glycoprotein I could interact with a non-collagenic microfibrillar component of subendothelium. The binding of factor VIII/von Willebrand factor to platelet membrane in the presence of ristocetin was decreased in the binding site for factor VIII/von Willebrand factor to allow platelet adhesion to subendothelium.

We have demonstrated that the specific determination and identification of plasma FbDP alone is not sufficient to follow the effectiveness of thrombolytic therapy. Some patients who received an intravenous infusion of rt-PA for myocardial infarction had very high plasma FbDP levels, although no recanalization was observed angiographically. Since in many cases the FbDP levels increased after recanalization, it is assumed that rethrombosis may occur during and after t-PA treatment.

An analysis of cell membrane material present in circulating immune complexes isolated from normal human sera by precipitation with polyethylene glycol (PEG) has been performed with the platelet alloantigen PlA1 and rhesus antigen used as markers. PEG precipitates obtained from the sera of subjects of known PlA1 and rhesus phenotype were resuspended in buffer and analyzed as representative of circulating immune complexes (CIC). The consumption of anti-PlA1 serum by CIC was determined by immunofluorescence and by inhibition of sodium 51chromate release from PlA1-positive target platelets. With these two techniques, PlA1 alloantigen activity was detected in CIC. This finding suggests that at least some of the cell membrane material present in CIC is derived from platelets.

The interaction of standard heparin and some low molecular weight heparin fragments (CY 222, mw 1,500-8,000 daltons) with human vascular endothelium in culture was studied using both 125I and 3H labeled ligands. A specific and saturable binding was shown for both labeled standard heparins. Two populations of binding sites for 3H-standard heparin could be distinguished: one of high affinity (KD = 0.12 microM), and another of lower affinity (KD = 1.37 microM). Total binding capacity was in the order of 10(7) molecules per cell. The same high level of affinity was calculated for unlabeled compounds from competition experiments with 125I-standard heparin. No other glycosaminoglycans, except a highly sulfated heparan (fraction IIA) could compete for heparin binding sites. A specific binding was also shown for 125I-CY 222. The affinity of unlabeled CY 222 was approximately ten times lower than that of unfractionated heparin. However, CY 222 could compete for approximatively 30% of standard heparin binding. Binding was not completely reversible. Even in the presence of a large excess of unlabeled compounds, a fraction of 25% of radioactive heparins remained bound to the endothelium. This fraction was three times lower if incubation was carried out at +4 degrees C, suggesting a possible incorporation of heparin into the endothelial cells.

The ultrastructure, cytochemistry, biochemistry, and functions of platelets from two patients with the familial gray platelet syndrome are described. Ultrastructural studies showed a lack of alpha-granules in the megakaryocytes in the vicinity of a marrow myelofibrosis and/or in platelets. A normal number of mitochondria and a slight increase of dense bodies was confirmed by labeling the whole patient with mepacrine. Platelet peroxidase (in the dense tubular system) and catalase-positive granules (revealed by the cytochemistry) were present. Platelets from both patients had severe deficiency of beta TG either after tritonization (less than 4% of normal) or thrombin treatment (less than 15% of normal), and the plasma beta TG levels were slightly increased. Functional studies of these platelets showed an uptake of [14C]5HT inhibited by reserpine similar to that in the control platelets. Thromboxane formation was within normal limits in the presence of arachidonic acid, ADP, collagen, or ionophore A23187, indicating that (1) the cyclooxygenase/thromboxane synthetase systems were not altered and (2) the phospholipase activities were not impaired. Although the platelet adhesion to prepolymerized fibrillar type III collagen and the ADP-, arachidonic acid-, and ionophore A23187-mediated aggregations were apparently normal, in every case the release of [14C]5HT was either at the low side of the normal range or decreased. This abnormality was increased when collagen or thrombin was added to either PRP or washed platelets from both patients, where at the same time, the aggregation and the release were markedly reduced. The results suggest that alpha-granules or their content has an influence on not only the platelet aggregation but also the dense bodies involved in the [14C]5HT release reaction.

The role of platelet plasma membrane in mediating cellular response to external stimuli was investigated by studying phosphatidylinositol turnover and structural physical molecular alterations. Phosphoinositide turnover was evaluated by phosphatidylinositol breakdown and phosphatidic acid (PA) synthesis. The membrane structure was investigated by measuring fatty acid chain organization, flexibility and movements using the spin label method. When platelets are stimulated by thrombin, a rapid phosphatidylinositol bisphosphate (PIP2) breakdown is observed, accompanied by an immediate PA synthesis. At the same time a membrane-bound calcium liberation within the cell is revealed by the decrease in chlortetracycline fluorescence, and the dense granule constituent serotonin is released from the cell. These events result in disorganization of the platelet membrane as estimated by the decrease in order parameter. All these thrombin-induced effects occur in the presence of EDTA, thus being independent of external calcium and aggregation. By contrast ionophore A 23187 does not induce any PIP2 breakdown nor structural disorganization and the dense granule release measured at 10 seconds is only very weak. These results were confirmed by studies on pathological platelets in which PIP2 breakdown was normal when release was normal, such as in Glanzman thrombasthenia, or did not occur when no granule release was measurable as in Gray-platelet syndrome. It is suggested that membrane-bound calcium has a structural rather than a functional role.

The effect of 3 prostaglandins (PG's) (I2, D2 and E1) on the adhesion of platelets to purified type III collagen has been investigated. A quantitative method for a specific evaluation of the adhesion has been applied and has revealed an inhibition of adhesion by low concentrations (10(-10)M) of PGs added before collagen; the effect varied as a function of the dose of PGs (maximum at 10(-6)M) which also induced an increase in the level of platelet cAMP. The inhibition of adhesion and the elevation of platelet cAMP followed the same time course and were either of short duration (rapid decrease in the induced effects after 15 and 45 seconds in the case of PGE1) or longer lasting (maximum effect maintained for 5 minutes in the case of PGI2 and D2). These effects were potentiated by a phosphodiesterase inhibitor such as theophylline (10(-3)M). The addition of PGs after collagen resulted in a reduction of the enhancement of cAMP, associated with a decrease in the inhibition of adhesion. Moreover, the addition of exogenous cAMP (dibutyryl N6-02' cAMP) induced a comparable inhibition. A correlation between the adhesion of platelets to collagen and the level of either endogenous or exogenous cAMP has been established. The PGs also inhibited the platelet release reaction from the alpha granules (beta TG) and the dense bodies. (5-HT and ADP). A greater inhibition of release than of adhesion was observed for the same doses of PGs added.

Washed human platelets have been incubated with the lectins WGA, ConA and RCA1, adsorbed to different-sized gold particles. Plasma membrane receptors for each lectin were then located by scanning and transmission electron microscopy.

The high incidence of thrombosis in inflammatory states and previous reports of increased adhesion of erythrocytes to endothelial cells in diabetes mellitus and sickle cell anemia prompted us to study the effect of fibrinogen and fibronectin on erythrocyte-endothelial interactions. Purified human fibrinogen enhanced erythrocyte adhesion in a concentration-dependent fashion. Erythrocytes from normal subjects, diabetics, and patients with sickle cell anemia were studied. The ratio between the adhesion of normal red cells in a 4 gm/L fibrinogen to adhesion in buffer without fibrinogen was 3.6 (p less than 0.001). Fibronectin also increased red cell adhesion but the effect was less than that of fibrinogen. The addition of fibronectin to fibrinogen limited the enhancing effect of fibrinogen, although the effect of the mixture was greater than that of fibronectin alone (p less than 0.05). Anti-von Willebrand factor and antifibronectin, which react with endothelial cells, also produced an increase in erythrocyte adhesion. The potentiation of adhesion by fibrinogen was also seen in experiments using red cells from patients with sickle cell anemia or diabetes mellitus. These observations provide possible mechanisms for the involvement of plasma proteins in vascular occlusive diseases.

We have previously shown that heparin was bound and internalized by cultured human endothelial cells. In this study, we have investigated the effect of heparin on endothelial cells growth. We found that heparin inhibited 3H-thymidine uptake as well as actual cell growth in a dose-dependent manner in the presence of low concentrations of human serum. Inhibition was maximal at 1% serum concentration and was abolished at 10%. Chasing experiments supported the role of membrane-bound heparin in this inhibition. Low molecular weight heparin fractions, or pentosan polysulfate, were equally effective in inhibiting 3H-thymidine uptake. On the other hand, the simultaneous addition of heparin and ECGS was synergic in stimulating 3H-thymidine uptake. These results suggest a modulatory role of heparin in endothelial cells growth.

Adult bovine aortic tissue was treated with 6 M guanidinium chloride in the presence of proteinase inhibitors to obtain an extract that was essentially devoid of collagenous components and appeared homogeneous by electron microscopy. When this extract was dispersed by sonication it was found to be a very potent inducer of human platelet aggregation. This interaction required the presence of von Willebrand factor and of its receptor (glycoprotein Ib) on platelet membrane. This was demonstrated by the fact that the aggregation of normal blood platelets resuspended in plasmas deficient in von Willebrand factor was significantly diminished as compared to aggregation in control plasma. Moreover, this aggregation was inhibited by a monoclonal antibody, IgG AN51, to platelet glycoprotein Ib. These studies provide direct biochemical evidence for the existence of a thrombogenic constituent of the vessel wall that is noncollagenous and von Willebrand factor-dependent.

The fibrin-mediated enhancement of the activation of plasminogen by tissue-type plasminogen activator observed with normal fibrin, is strongly decreased with fibrin Dusard, although the binding of tissue-type plasminogen activator to this fibrin is normal. This impaired fibrin-mediated plasminogen activation is most likely related to the history of recurrent thrombosis and pulmonary embolism observed in this family.

Platelet function, factor VIII, fibrinogen levels, and fibrinolysis were studied in Europeans and in two groups of Nigerians living in Zaria, northern Nigeria, in order to see whether differences could help to explain the low incidence of atheroma and thrombosis in Nigerians. We confirmed the relative thrombocytopenia and observed a rapid disaggregation after ADP-induced platelet aggregation in Nigerians. The most striking difference was a reduced or absent ristocetin-induced platelet aggregation in Nigerian platelet-rich plasma, probably due to a plasma component interacting with the von Willebrand activity (VWF), since factor VIII coagulant activity, factor VIII related antigen, and isolated VWF were normal or high by European standards. Group II (rural population), but not group I (senior university staff in Zaria) of the Nigerians, tended to have high serum fibrinogen concentrations. Spontaneous fibrinolytic activity was enhanced in most Nigerians compared to the Europeans and was normally increased after venostasis in proportion to the initial activity. Fibrinolysis and ristocetin-induced platelet aggregation values for the Nigerians in group I were intermediate between European and Nigerian in the group II values, suggesting that differences were due more to environmental than to genetic factors.Relative thrombocytopenia, disaggregation after ADP-induced aggregation, inhibition of ristocetin-induced platelet aggregation, and active fibrinolysis help to explain the infrequency of thrombotic disease in Africans. Also the low incidence of atheroma may follow from less platelet adherence and less platelet release of mitogenic factors, which cause intimal hyperplasia.

The relation between blood glucose control and erythrocyte adhesion to cultured human vascular endothelial cells was studied in 12 Type 1 (insulin-dependent) diabetic patients. Erythrocyte adhesion was measured before, 8 days and 6 weeks after continuous subcutaneous insulin infusion (CSII). Compared to controls, erythrocyte adhesion expressed as an adhesion ratio was increased in 10 diabetic patients at the first examination (adhesion ratio greater than 1.52). After 8 days, adhesion ratio was normalized in 5 patients. After 6 weeks, adhesion ratio was normal in 7 patients and significantly lower in the group of 12 patients (p less than 0.01). The adhesion ratio was significantly correlated with glycosylated haemoglobin values (p less than 0.001). Insulin did not directly affect erythrocyte adhesion. Adequate insulin treatment modifies erythrocyte adhesion through a metabolic effect which needs longer than 1 week to be effective.

Bernard-Soulier syndrome is a constitutional thrombopathy with an impaired platelet adhesion to the vessel wall. Since the first description in 1948 many works had been reported, and recently a molecular abnormality of the platelet membrane was shown. Interactions between specific membrane sites and platelet functions are now proposed.

Endothelial cell alterations are considered to be a primary event in thrombosis and atherosclerosis. The adhesion of red cells from diabetic patients or patients with sickle cell anemia was measured using endothelial cell culture and a radiometric technique. The adhesion was significantly increased (P less than 0.001). Ultrastructure observation of red cells adhering to endothelial cells showed endothelial cell deformations, membrane modifications and formation of an anchoring device. Morphological changes support the hypothesis of an endothelial cell stimulation or damage during adhesion already suggested by the increased prostacyclin release during red cell adhesion.

Crossed immunoelectrophoresis of "Triton X-100" solubilized proteins from (125I) iodinated normal and abnormal (Bernard-Soulier and thrombasthenic) platelets was performed using rabbit antibodies raised against whole, normal platelets. In Bernard-Soulier platelets neither of the two surface oriented forms of GP I, glycocalicin and glycoprotein Ib (band 13) were detected. In two patients with type I thrombasthenia, the most prominent precipitate (No. 16) and platelet fibrinogen (No 24) were not detected whereas in one patient with type II thromblasthenia, fibrinogen was normally detected and the amount of protein 16 was around 15% of the normal level. The alloantibody IgG L..., able to induce a "thrombasthenic-like" reactivity in normal human platelets, has been shown to be directed against protein 16.

Originally described in vascular endothelial cells, Weibel-Palade bodies were considered as specific of this cellular type, as they have never been reported elsewhere. Weibel-Palade bodies serve as storage granules for von Willebrand factor which is stored in microtubular form. Besides endothelial cells von Willebrand factor is also synthetized by bone marrow megakaryocytes. Von Willebrand factor has been located in alpha-granules of megakaryocytes and blood platelets. We describe true Weibel-Palade bodies in pig megakaryocytes, and also alpha-granules which look like an evolutionary form of Weibel-Palade bodies. Von Willebrand Factor is most likely stored in microtubular form in these two types of structure. This is supported by the absence of microtubules in these granules in cells obtained from pigs homozygous for the von Willebrand disease (lacking totally this protein).

Activation of plasminogen by tissue-type plasminogen activator (tpA) is potentiated by fibrin. We have demonstrated the role of fibrin polymerization in the potentiating effect of tpA-induced fibrinolysis. Therefore a pathogenic mechanism of thrombotic disorder may be related to an abnormal fibrin polymerization: the abnormal clot being less accessible to fibrinolysis than normal one. This defective lysis may be due to a defective enhancement by the abnormal fibrin of plasminogen activation by tpA, as demonstrated for fibrinogen Dusard, a congenital dysfibrinogenemia associated with a very severe thrombotic disorder. In some other cases, a decrease in the availability of the plasmin cleavage sites in fibrin clot may be involved. On the contrary, some antithrombotic drugs such as pentosane polysulfate in modifying clot structure allow a better degradation of fibrin clot by fibrinolytic enzymes. It is speculated that this enhanced fibrinolysis could explain, almost in part, the antithrombotic action of these drugs.

Platelets and intravascular coagulation are involed in the pathogenesis of renal allotransplant rejection phenomena: fibrin deposition is a prominent feature of hyperacute rejection; arteriolar microthromboses when they are present, worsen the prognosis of an acute reversible rejection; the organisation of platelets and fibrin deposits on the intima may lead to the "endarteritis obilterans" of chronic bascular rejection. The interactions between endothelium, platelets, coagulation, fibrinolysis and cellular or humoral effectors of the immune respone were studied and special reference was made to their role in vascular rejection. The poor prognosis in vasuclar lesions justifies therapeutic trials with drugs inhibiting the early events of thrombogenesis.

Previous studies have shown that the receptor for quinine- and quinidine-dependent antibodies is not expressed on the surface of platelets from patients with the Bernard-Soulier (B-S) syndrome. We now report data to suggest that these platelets lack the receptor for these antibodies. Since B-S platelets are also missing 2 related components of the GP I complex, GP lb and glycocalicin, our findings suggest that the receptor for quinine- and quinidine-dependent antibodies may be associated with this complex on normal platelets. An antibody previously shown to be directed against a surface antigen that migrated in the GP I position on SDS-polyacrylamide gel electrophoresis specifically blocked the reaction of the receptor with quinine- or quinidine-dependent antibodies. Purified glycocalicin, however, lacked detectable receptor activity. In contrast, a mixture of GP lb and a putative structural analog of this glycoprotein (Mr 210,000) eluted from wheat germ affinity columns after chromatography of Triton X soluble preparations of platelets or membranes was shown to contain at least 80% of the total receptor activity. Our results strongly suggest that the receptor is associated with GP lb and/or its high m.w. structural analog(s).

Prothrombinase activities of platelets have been measured in diluted platelet-rich plasma using a chromogenic substrate assay and purified coagulation factors. No abnormalities in prothrombinase activities were found for platelets from patients with storage pool disease (dense-body deficiency), grey platelet syndrome, and Glanzmann's thrombasthenia. It is concluded that neither release of dense bodies and alpha-granules nor aggregation of platelets are essential prerequisites for exposure of a procoagulant surface. Platelets from patients with Bernard-Soulier syndrome, however, have approximately 10-fold higher prothrombinase activities in the non-stimulated form than normal non-stimulated platelets. The increased procoagulant activity cannot be completely ascribed to an increase in platelet size. It is suggested that the increased prothrombinase activity reflects an increased exposure of phosphatidylserine at the outer surface of non-stimulated Bernard-Soulier platelets, earlier described by Perret et al (1983).

A decreased platelet adhesion to rabbit aorta subendothelium (Baumgartner technique) is confirmed in the Bernard Soulier (giant platelet) syndrome. Electron microscope techniques using a purified antibody against Factor VIII/von Willebrand protein, revealed an apparently normal presence of the Factor VIII/von Willebrand protein on the Bernard Soulier platelets. Electrophoretic characterization of the major protein and glycoprotein components of the Bernard Soulier platelets following sodium dodecyl sulfate solubilization indicated a relatively normal protein content but suggested a reduced content of the 155,000 molecular weight major platelet glycoprotein. This was confirmed by a reduced release of high molecular weight acidic glycopeptides following incubation of washed Bernard Soulier platelets with trypsin. It is proposed that this abnormality may be related to the previously reported reduced sialic acid content and the reduced electrophoretic mobility of the Bernard Soulier platelets and that a glycoprotein reduced or abnormal in the Bernard Soulier platelets is necessary for the normal adhesion of platelets to subendothelium.

This paper reports the biological data found during an acquired bleeding disorder occurring in a 65-year-old woman affected with chronic lymphocytic leukaemia. They consists of haemostatic abnormalities which resemble an presently undescribed association of an acquired von Willebrand's syndrome with a thrombopathy. von Willebrand abnormalities were temporarily corrected by infusion of normal cryoprecipitate and reproduced in vitro by the incubation of normal platelets with the patient's IgA. The platelet defect was partially corrected after corticotherapy. The possible relationship of the associated defects is discussed.

We have measured the binding of 14C-ADP to isolated human platelet membranes by a technique using 0.8-mum Millipore filters to separate unbound tracer from membrane-bound nucleotide. The binding was dependent on the time of incubation and on the nucleotide concentration in the medium. The affinity constant was found to be comprised between 0.35 x 10(6) and 0.55 x 10 (6) M-1. Platelet membranes prepared from different thrombasthenic patients bound 14C-ADP with the same kinetic parameters as those from normal subjects. The affinity constant as determined for two of these thrombasthenic platelet membrane preparations was in the normal range.

Hypercalcemia was associated with osteolytic bone lesions in a 60-year-old woman with chronic myelogenous leukemia in the accelerated phase. Using highly specific antisera to parathyroid hormone, radioimmunoassays disclosed elevated levels of carboxyl-terminal (53-84) and intermediate (44-68) fragments. In addition, concomitant variations of serum calcium level and leukocyte counts, increased urinary c-AMP excretion, morphological integrity of parathyroid glands, and absence of bone resorbing activity in myeloblast culture supernatants are consistent with the hypothesis that the humoral hypercalcemia was due to the excessive production of PTH. This production may have been ectopic, although no PTH secretion was demonstrated in myeloblast culture supernatants.

Platelets from patients with the gray platelet syndrome have decreased recognizable alpha granules and are markedly deficient in some alpha-granule secretory proteins. Using immunocytochemical techniques with antibodies to an alpha-granule membrane protein, GMP-140, we identified the membranes of intracellular vesicles in gray platelets as alpha-granule membranes. Gray platelets contained normal amounts of GMP-140 as measured by electroimmunoassay. The activation of gray platelets with thrombin caused GMP-140 to be redistributed to the plasma membrane surface, as in normal platelets. In agreement with previous studies, an endogenously synthesized secretory protein, platelet factor 4, was undetectable in gray platelets. However, the alpha-granule proteins albumin and IgG, which are thought to be derived from endocytosis of plasma proteins into megakaryocytes, were present in substantial quantities and were secreted efficiently from gray platelets. Therefore, the fundamental defect in the gray platelet syndrome may be in the targeting of endogenously synthesized secretory proteins to developing alpha granules in megakaryocytes.

A young female patient with Gardner's syndrome developed post-transfusion purpura. Serological studies demonstrated the presence of an antibody directed not against PlA1 but against a new antigen, Leka. This is the first case of post-transfusion purpura not associated with immunization against the PlA1 antigen. The Leka antigen is present in 98% of the French population. Its absence on platelets from patients with thrombasthenia suggests that it is located on the IIb IIIa platelet glycoprotein complex.

The expression of the von Willebrand factor (vWF) gene by cultured endothelial cells from the porcine pulmonary artery, aorta, and lung was compared at the levels of messenger (m)RNA and antigen. Steady-state levels of vWF mRNA were determined by dot-blot analysis using a partial human vWF cDNA as the hybridization probe; vWF mRNA from cultured aortic endothelial cells, and vWF antigen secreted into the culture supernatants were barely detectable. In contrast, vWF mRNA and antigen from the pulmonary artery endothelial cells were approximately eight to nine times that demonstrated by aortic cells. Levels of vWF mRNA and antigen in cultured lung cells were intermediate of those found in the pulmonary artery and aorta and correlated with the estimated number of cells demonstrated to be of endothelial origin in the mixed cell populations grown from the lung. Differences between the levels of vWF mRNA found in cultured cells from the pulmonary artery and those found in the aorta were maintained in cells processed directly from these vessels. Correlation between the levels of vWF mRNA and antigen in endothelial cells from different vessels of the pig suggests that the differential control of vWF synthesis is at the level of transcription. Furthermore, maintenance in cultured cells of the difference in transcription rates that were observed in vivo suggests that vWF gene expression is not exclusively regulated through environmental factors.

Two unrelated patients with "pseudo" ("platelet-type")-von Willebrand's disease (vWD) are described demonstrating thrombocytopenia with a prolonged bleeding time, ristocetin-induced platelet aggregation at low stimulus concentrations, decreased levels of ristocetin-cofactor activity (vWF:RCo), slight cryoprecipitate-induced platelet aggregation in the absence of ristocetin, and a lack of the high molecular weight factor VIII-von Willebrand factor (FVIII/vWF) multimers in plasma. Isolated washed patient platelets bound more FVIII/vWF at high (1 and 0.75 mg/ml) and low (0.5 and 0.25 mg/ml) ristocetin concentrations than control platelets. Fresh or paraformaldehyde-fixed washed platelets from these patients also bound more specific monoclonal antibody to glycoprotein Ib (25,000 binding sites per cell) than normal platelets (15,000 +/- 3,300 binding sites per cell). Results obtained in control patients with thrombocytopenia and increased platelet size (May-Hegglin anomaly and vWD type IIB) excluded a nonspecific increase of glycoprotein Ib in the platelets of the patients with pseudo-vWD. These data indicate that in pseudo-vWD, the primary abnormality lies in the platelet and is related to a quantitative and/or qualitative anomaly of platelet membrane glycoprotein Ib.

Platelet GP IIb-IIIa complex is missing or strongly reduced in thrombasthenia type I or II; in parallel the binding of fibrinogen is either nil or strongly reduced after platelet activation and these platelets do not aggregate. In the platelets of the described variant, GP IIb-IIIa level and PLA1 antigen are around 50% of the normal but the fibrinogen sites are either missing or unavailable. The hypothesis is that the patient's platelets lack the specific receptor site for fibrinogen at the GP IIb-IIIa level. However, platelet fibrinogen is normal as is the clot retraction. The study of this variant allows new concepts on platelet aggregation and clot retraction.

To investigate the platelet contribution to the development of myelofibrosis in hairy cell leukaemia (HCL), we have studied two platelet alpha granule components in 15 patients with HCL before chemotherapy: mitogenic activity was measured by 3H thymidine incorporation in BALB/C 3T3 cells and beta thromboglobulin (beta TG) assayed by radioimmunoassay (RIA). Platelet mitogenic activity and beta TG content were significantly decreased in the patients as compared to the control subjects. The nine patients who were treated with recombinant human interferon (IFN alpha A) were restudied after 4 months of therapy. The levels of both mitogenic activity and beta TG platelet content were significantly increased after IFN alpha-treatment with a complete response in five of the nine treated patients, a partial response in two and no response in the two others. HCL seemed therefore to be responsible for an acquired platelet alpha granule defect. As in the grey platelet syndrome a relationship between this abnormal platelet granule storage and the development of myelofibrosis is suggested in HCL.

We have studied the fibrinogen binding to the platelets of four type I thrombasthenic two type II thrombasthenic patients and 15 normal subjects. The amount of [125I]fibrinogen bound to washed platelets in the presence or absence of 10 micrometers ADP was measured and the results expressed as the percentage of the total radioactivity added. The mean value for specific binding to the platelets of normal subjects was 3.7 (range 1--7.25) at 15 min after the addition of ADP. Type I thrombasthenic patients lack glycoproteins (GP) IIb and IIIa in the platelet membrane and intra-platelet fibrinogen. In all four patients studied, little or no specific fibrinogen binding was detected. Platelets of type II thrombasthenic patients contain detectable, although markedly reduced amounts of GP IIb and IIIa, and subnormal or normal levels of fibrinogen. In these platelets, significant specific fibrinogen binding was observed, the values being slightly below or at the lower end of the normal range. Fibrinogen binding to normal platelets was also measured after the addition of an IgG purified from the serum of a multi-transfused thrombasthenic type I patient. This antibody was previously shown to be directed against the GP IIb/IIIa complex as located by crossed immunoelectrophoresis. Specific fibrinogen-binding was blocked and the extent of the inhibition was directly proportional to the concentration of the antibody added.

Platelet aggregation of an afibrinogenaemic patient's platelet rich plasma (PRP) was greatly decreased when ADP was used for stimulation. In the presence of collagen or arachidonic acid the changes in light transmission recorded during platelet aggregation of patient's PRP were similar to those observed with normal PRP but the size of aggregates appeared to be smaller in comparison with those observed with normal platelets. In addition, thrombin-induced aggregation of washed platelets was similar to normal platelets. The interpretation was made possible because the fibrinogen level in plasma and in platelets was found to be almost nil as demonstrated by both an Elisa procedure described here and the determination of fibrinopeptide A (fpA). Furthermore, fibrinogen fragments, which could result from abnormal synthesis and therefore replace fibrinogen in platelet aggregation, were undetectable by immunological analysis using specific antibodies against A alpha, B beta and gamma chains and 10 different monoclonal antibodies against fibrin degradation products.