Diagnostic challenges in von Willebrand disease. Report of two cases with emphasis on multimeric and molecular analysis.

Identification of qualitative variants of von Willebrand disease (VWD) can be a diagnostic challenge because of discrepant results obtained in the multiple laboratory tests available for its appropriate classification. We report two cases of infrequent inherited variants of VWD with unclear preliminary results with the test panel available at the time of first consultation and that were finally diagnosed as a VWD type 2A/IID with a c.8318 G > C, p.Cys2773Ser mutation and a VWD type 2M with c.4225 T > G, p.Val1409Phe mutation, respectively. The description of these two cases highlights that despite the limited diagnostic panel for the evaluation of von Willebrand Factor (VWF) functionality, the multimeric analysis and genetic family studies were fundamental tools to achieve the final diagnosis.

Reduced ADAMTS13 activity is associated with thrombotic risk in systemic lupus erythematosus.

BACKGROUND: Severe deficiency of ADAMTS13 activity leads to von Willebrand factor (VWF) ultralarge multimers with high affinity for platelets, causing thrombotic thrombocytopenic purpura. Other pathological conditions with moderate ADAMTS13 activity exhibit a thrombotic risk. We examined the ADAMTS13 activity in systemic lupus erythematosus (SLE) and its value as a thrombotic biomarker. METHODS: ADAMTS13 activity, VWF antigen and multimeric structure, and vascular cell adhesion molecule 1 (VCAM-1) were measured in plasma samples from 50 SLE patients and 50 healthy donors. Disease activity (systemic lupus erythematosus disease activity index; SLEDAI) and organ damage (systemic lupus international collaborating clinics) scores, thrombotic events, antiphospholipid syndrome (APS) and antiphospholipid antibodies (aPLs) were registered. RESULTS: SLE patients showed decreased ADAMTS13 activity and high VWF levels compared with controls (66 ± 27% vs. 101 ± 8%, P < 0.01, and 325 ± 151% vs. 81 ± 14%, P < 0.001). VCAM-1 levels were higher in SLE patients (P < 0.05). Considering three groups of SLE patients depending on ADAMTS13 activity (>60%, 60-40% and <40%), comparative analysis showed significant association between ADAMTS13 activity and SLEDAI (P < 0.05), presence of aPLs (P < 0.001), APS (P < 0.01) and thrombotic events (P < 0.01). Reduced ADAMTS13 activity together with increased VWF levels were especially notable in patients with active disease and with aPLs. CONCLUSION: ADAMTS13 activity, in combination with other laboratory parameters, could constitute a potential prognostic biomarker of thrombotic risk in SLE.

Role of fibrinogen concentrates for treatment of critical perioperative hemorrhage.

Acquired hypofibrinogenemia is a frequent cause of maintained bleeding in perioperative high-risk settings. Loss, consumption and dilution under resuscitation fluid therapy are the principal causes for fibrinogen depletion. Severe hypofibrinogenemia is frequently associated with an early bleeding complication that cannot be reliably avoided with high-ratio plasma transfusion strategies. Real-time monitoring with viscoelastic hemostatic assays is a useful tool for timely diagnosis and treatment of detected coagulopathies. Replenishment of fibrinogen in uncontrolled bleeding events is currently recommended by most published guidelines, suggesting treatment thresholds to maintain a minimum of 1.5 g/L plasma fibrinogen concentration for nonobstetrical hemorrhage. Fibrinogen concentrates, originally licensed for treatment of bleeding episodes in patients with congenital hypo-, dys- or afibrinogenemia disorders, are used in many clinical situations as supplementary therapy for the treatment of acquired hypofibrinogenemia. This review seeks to provide an overview of the most relevant topics associated to fibrinogen replacement therapy for critical perioperative hemorrhage highlighting currently available evidence on the risk/benefit profile of purified fibrinogen concentrates for this extended clinical indication.

The secretory mechanisms in equine platelets are independent of cytoskeletal polymerization and occur through membrane fusion.

Studies in animal models are useful to understand the basic mechanisms involved in hemostasis and the functional differences among species. Ultrastructural observations led us to predict differences in the activation and secretion mechanisms between equine and human platelets. The potential mechanisms involved have been comparatively explored in the present study. Equine and human platelets were activated with thrombin (0.5 U/ml) and collagen (20 µg/ml), for 90 seconds, and samples processed to evaluate: i) ultrastructural changes, by electron microscopy, ii) actin polymerization and cytoskeletal assembly, by polyacrylamide gel electrophoresis, and iii) specific molecules involved in activation and secretion, by western blot. In activated human platelets, centralization of granules, cytoskeletal assembly and fusion of granules with the open canalicular system were observed. In activated equine platelets, granules fused together forming an organelle chain that fused with the surface membrane and released its content directly outside the platelets. Human platelets responded to activation with actin polymerization and the assembly of other contractile proteins to the cytoskeleton. These events were almost undetectable in equine platelets. When exploring the involvement of the synaptosomal-associated protein-23 (SNAP-23), a known regulator of secretory granule/plasma membrane fusion events, it was present in both human and equine platelets. SNAP-23 was shown to be more activated in equine platelets than human platelets in response to activation, especially with collagen. Thus, there are significant differences in the secretion mechanisms between human and equine platelets. While in human platelets, activation and secretion of granules depend on mechanisms of internal contraction and membrane fusion, in equine platelets the fusion mechanisms seem to be predominant.

Apigenin inhibits platelet adhesion and thrombus formation and synergizes with aspirin in the suppression of the arachidonic acid pathway.

Previous studies using washed platelets demonstrated that certain flavonoids inhibit platelet function through several mechanisms including blockade of TxA(2) receptors (TPs). We aimed to analyze the binding capacity of flavonoids to TPs in platelet rich plasma (PRP), investigated their effect in flowing blood, and evaluated the ability of apigenin to improve the efficacy of aspirin in the inhibition of platelet aggregation. The binding of flavonoids to TPs in PRP was explored using binding assays and the TP antagonist [ (3)H]SQ29548. Effects of flavonoids on platelet adhesion were assessed using arterial subendothelium with annular plate perfusion chambers, and global evaluation of apigenin on high-shear-dependent platelet function was determined by the PFA-100. To evaluate the ability of apigenin to potentiate the effect of aspirin, arachidonic acid-induced platelet aggregation was measured prior to and after consumption of subaggregatory doses of aspirin in the presence or absence of apigenin. Binding assays revealed that apigenin was an efficient competitor of [ (3)H]SQ29548 binding to PRP ( K i = 155.3 +/- 65.4 microM), and perfusion studies showed that apigenin, genistein, and catechin significantly diminished thrombus formation when compared to control (26.2 +/- 3.8, 33.1 +/- 5.2, and 26.2 +/- 5.2 vs 76.6 +/- 2.6%, respectively; p < 0.05). Apigenin, similarly to the TP antagonist SQ29548, significantly prolonged collagen epinephrine-induced PFA-100 closure time in comparison to the control and, when added to platelets that had been exposed in vivo to aspirin, potentiated its inhibitory effect on platelet aggregation. The inhibitory effect of some flavonoids in the presence of plasma, particularly apigenin, might in part rely on TxA(2) receptor antagonism. There is a clear increase in the ex vivo antiplatelet effect of aspirin in the presence of apigenin, which encourages the idea of the combined use of aspirin and certain flavonoids in patients in which aspirin fails to properly suppress the TxA(2) pathway.

Emicizumab for routine prophylaxis to prevent bleeding episodes in patients with hemophilia A.

Hemophilia A is an X-linked bleeding disorder caused by defects in the gene encoding factor VIII (FVIII). Routine prophylaxis with exogenous FVIII requires frequent intravenous injections. One of the most challenging issues in the treatment of hemophilia A is the development of alloantibodies against infused FVIII. Presence of inhibitors results in an ineffective factor replacement therapy and increases the risk of morbidity and mortality in these patients. Therefore, there is growing interest in the development of new strategies for the prophylaxis and prevention of bleeding in patients with hemophilia to circumvent these drawbacks. Emicizumab (ACE-910; Roche, Genentech and Chugai Pharmaceutical) is a recombinant humanized bispecific antibody that restores the function of missing FVIII by bridging activated FIX and FX, simulating the cofactor function of FVIII.

Betrixaban: a direct oral inhibitor of activated factor X for the prophylaxis of venous thromboembolism in patients hospitalized for acute medical illness.

Venous thromboembolism (VTE), comprising deep vein thrombosis and pulmonary embolism, is a serious clinical and public health concern. Hospitalization is a major risk factor for developing VTE. Hospital-associated events account for more than 50% of all cases of VTE. Heparins have demonstrated to be efficacious in the prevention of VTE in medically ill patients. Despite the demonstrated efficacy and safety of the available direct oral anticoagulants in the prevention and treatment of different thromboembolic conditions, their net benefit in the prevention of VTE in hospitalized medically ill patients has not been fully confirmed. Betrixaban is an oral, specific and direct inhibitor of human coagulation factor Xa with demonstrated efficacy and safety for the prevention of VTE in patients undergoing total knee replacement and in patients with nonvalvular atrial fibrillation. Recent studies have successfully evaluated betrixaban 80 mg once daily in the prevention of VTE in acute medically ill patients in a large phase III trial. This review will address preclinical pharmacology and main aspects of the clinical development of betrixaban as an antithrombotic agent, with specific attention to recent studies on the prophylaxis of VTE in a specific population of patients hospitalized for acute medical illnesses.

Andexanet alfa: a recombinant mimetic of human factor Xa for the reversal of anticoagulant therapies.

Activated coagulation factor X (FXa) is a common target for classic and newer anticoagulants. Parenteral anticoagulants with an indirect inhibitory action on FXa (low-molecular-weight heparins) have a well-established clinical efficacy in the prophylaxis and therapy of thromboembolic conditions. More recently developed direct oral anticoagulants (DOACs) have emerged as a new class of antithrombotic drugs. Rivaroxaban, apixaban and edoxaban are direct inhibitors of FXa approved for the management of venous thromboembolism and stroke prevention in atrial fibrillation. Although these DOACs are associated with fewer hemorrhagic side effects than classic vitamin K antagonists, bleeding is still a main complication. FXa antagonists had no specific agents that could reverse their antihemostatic effects. Andexanet alfa is a modified, recombinant human FXa molecule with an enhanced ability to bind to both direct and indirect FXa inhibitors, but unable to contribute to blood coagulation mechanisms. Andexanet alfa is designed to reverse the anticoagulant effects of FXa inhibitors. This review will address the preclinical pharmacology and the main aspects of the clinical development of andexanet alfa for the reversal of anticoagulant therapies with an inhibitory action on FXa. It will also summarize additional completed or ongoing studies on andexanet alfa available to the scientific community until present.

Endothelial damage is aggravated in acute GvHD and could predict its development.

The aim of the present study was to explore whether there is enhanced endothelial dysfunction in patients developing acute GvHD (aGvHD) after allogeneic hematopoietic cell transplantation (allo-HCT) and to identify biomarkers with predictive and/or diagnostic value. In in vitro experiments, endothelial cells (ECs) were exposed to serum from patients with (aGvHD, n=31) and without (NoGvHD, n=13) aGvHD, to evaluate changes in surface adhesion receptors, the reactivity of the extracellular matrix by measuring the presence of Von Willebrand factor (VWF) and platelet adhesion, and the activation of intracellular signaling proteins. Plasma levels of VWF, ADAMTS-13, TNF receptor 1 (TNFR1), soluble vascular cell adhesion molecule 1 and soluble intercellular adhesion molecule 1 were also measured. In vitro results showed a more marked proinflammatory and prothrombotic phenotype in ECs in association with aGvHD. Regarding circulating biomarkers, levels of VWF and TNFR1 above an optimal cutoff score, taken independently or combined, at day 7 after allo-HCT, would be able to positively predict that around 90% of patients will develop aGvHD. Our results demonstrate that endothelial damage is aggravated in those allo-HCT recipients developing aGvHD, and that VWF and TNFR1 are promising predictive aGvHD biomarkers. These findings could contribute to improve the understanding of the pathophysiology of aGvHD.

Endothelial damage in major depression patients is modulated by SSRI treatment, as demonstrated by circulating biomarkers and an in vitro cell model.

There is a link between depression, cardiovascular events and inflammation. We have explored this connection through endothelial dysfunction, using in vivo and in vitro approaches. We evaluated circulating biomarkers of endothelial dysfunction in patients with major depression at their diagnosis (MD-0) and during antidepressant treatment with the selective serotonin reuptake inhibitor escitalopram, for 8 and 24 weeks (MD-8 and MD-24). Results were always compared with matched healthy controls (CON). We measured in vivo circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) in blood samples, and assessed plasma levels of soluble von Willebrand factor (VWF) and vascular cell adhesion molecule-1 (VCAM-1). CEC counts, soluble VWF and VCAM-1 were statistically elevated in MD-0 (P<0.01 versus CON) and gradually decreased during treatment. Conversely, EPC levels were lower in MD-0, tending to increase throughout treatment. In vitro studies were performed in human endothelial cells cultured in the presence of sera from each study group. Elevated expression of the inflammation marker intercellular adhesion molecule-1 and oxidative stress, with lower presence of endothelial nitric oxide synthase and higher reactive oxygen species production, were found in cells exposed to MD-0 sera (P<0.05 versus CON). These results were normalized in cells exposed to MD-24 sera. Thrombogenicity of extracellular matrices generated by these cells, measured as expression of VWF, tissue factor and platelet reactivity, showed non-significant differences. We provide a model of cultured endothelial cells reproducing endothelial dysfunction in naive patients with major depression, demonstrating endothelial damage and inflammation at diagnosis, and recovering with selective serotonin reuptake inhibitor treatment for 24 weeks.

Differences and similarities in tyrosine phosphorylation of proteins in platelets from human and pig species.

BACKGROUND: Pigs have been widely used as animal models to study hemostasis. However, there are significant differences when comparing the hemostatic behavior of pig and human platelets. OBJECTIVE: To investigate signaling through tyrosine-phosphorylation of proteins in pig platelets after activation in suspension or by adhesion under flow conditions, in comparison with human platelets. METHODS: Activation of platelet suspensions was performed with thrombin (T; 0.1 and 1 U mL(-1)) and type I collagen (Col-I; 20 microg mL(-1)), at two different time points (30 and 90 s). Activation by adhesion was carried out on Col-I-coated coverslips, using citrated whole blood samples perfused through a parallel-plate chamber. RESULTS AND CONCLUSIONS: Significant differences between pig and human platelets were detected before and after activation. Activation of pig platelets required higher concentrations of thrombin, as well as increased activation times, to achieve similar levels of tyrosine phosphorylation. Proteins p160, p140, p85 and pp62, present in human platelets, were not detected in profiles corresponding to activated pig platelets. A protein of 70 kDa appeared only in pig platelet profiles, p55 was highly phosphorylated, and the phosphorylation levels of some proteins were significantly different from those found in human platelet profiles. In profiles corresponding to adhered pig platelets, p85 and p62 were absent, and p115 appeared highly phosphorylated. As observed in suspension studies, p70 and p55 appeared specifically in adhered pig platelets. Our study shows that the phosphotyrosine proteins involved in the activation of pig platelets are significantly different from those observed in activated human platelets. These findings may help to explain the differing adhesive and cohesive properties of platelets from both species, which should be considered when extrapolating results.

Erythropoietin improves signaling through tyrosine phosphorylation in platelets from uremic patients.

Erythropoietin has shown to be effective in the correction of the hemostatic defect present in uremic patients. We have investigated the possible effect of recombinant human erythropoietin (rHuEPO) on the signaling processes occurring in platelets. Platelet suspensions were obtained from hemodialyzed patients before and after at least one month of initiating treatment with rHuEPO. Aliquots of non-activated or thrombin-activated platelets were treated to obtain platelet lysates or processed to extract platelet cytoskeleton. Samples were resolved by 8% SDS-polyacrylamide gel electrophoresis followed by Western blotting. After thrombin activation, proteins p120, p85, p78, p75, pp62, pp60, p59, p58, p56, p54 and p52 associated with the Triton-insoluble cytoskeletal fraction appeared phosphorylated in control profiles. In profiles from platelets obtained from uremic patients before treatment with rHuEPO, only proteins p58 and p56 appeared clearly and p54 was slightly phosphorylated. However, in platelets from the same patients under rHuEPO treatment, thrombin-induced phosphorylation improved to levels even above those observed in control profiles. Specially, the band at 54KDa appeared consistently more phosphorylated in all the patients under rHuEPO treatment. Although it is accepted that part of the hemostatic effect of erythropoietin is mediated by an increase in hematocrit, our study suggests that it enhances platelet signaling in uremic platelets which may explain the improvement of platelet response to activating stimulus before clinically noticeable elevation of hematocrit.

Desmopressin (DDAVP) enhances platelet adhesion to the extracellular matrix of cultured human endothelial cells through increased expression of tissue factor.

The effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DDAVP-treated ECMS were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin, 20 U/ml). Perfusions with run for 5 min at a shear rate of 800 s-1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p < 0.05 and p. < 0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p < 0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

Uremic medium disturbs the hemostatic balance of cultured human endothelial cells.

We have investigated the ability of serum from uremic patients to modify the thrombogenic properties of the endothelium. The effects of uremic medium on the morphology of endothelial cells (ECs), and their resistance to flow was analyzed. The influence of uremic media on the reactivity of the extracellular matrix (ECM) generated by ECs towards normal platelets was evaluated in a parallel-plate perfusion chamber. Exposure of ECs to uremic medium resulted in abnormal cell morphology and signs of an accelerated growth. Detachment of ECs exposed to circulating blood was increased when cells had been grown with media supplemented with uremic serum (21% vs. 14% non exposed). Platelet deposition was significantly elevated on ECMs generated in the presence of uremic media (uremicECMs) (p<0.01 vs. control studies). Effects of uremic serum were not observed at short incubation periods (5 h) but were evident after 24 or 72 h of incubation. Northern blot analysis revealed increased expression of tissue factor (TF) mRNA in ECs exposed to uremic conditions. Immunocytochemical methods detected an augmented expression of TF antigen on uremic ECMs. Incubation of ECMs with an antibody to human tissue factor prevented the increase in platelet deposition observed in uremic ECMs, suggesting that the presence of TF in ECM could be responsible for the enhanced platelet deposition. Results from our study indicate that uremic medium impairs the antithrombotic functions of cultured endothelial cells.

New developments in thrombosis therapy.

Researchers presenting at the 16th International Congress on Thrombosis (Mediterranean League against Thromboembolic Disease), held May 58, 2000, in Porto, Portugal, reviewed the latest developments in the treatment of thrombosis. Several symposia focused attention on the new indications for low-molecular-weight heparins and provided updates on trials. The roles of tissue factor and tissue factor pathway inhibitor were extensively reviewed, and a number of late-stage developmental anticoagulants were presented.

State-of-the-art knowledge on thrombosis and hemostasis.

The XVIII Congress of the International Society on Thrombosis and Haemostasis, held July 6-12, 2001, in Paris, France, covered the latest advances in the field of thrombosis and hemostasis, including vascular biology. Plenary conferences, state-of-the-art lectures, symposia, and oral and poster presentations focused most of the attention on the current research on the pathogenesis and treatment of thrombotic and hemorrhagic disorders and provided updates on trials currently under way or already completed.

Latest advances from basic and clinical research in hematology.

New treatments in hematological malignancies were a focal point of sessions and presentations at the 42nd Annual Meeting of the American Society of Hematology, held December 15, 2000, in San Francisco, California, U.S.A. The meeting also provided discussion on pathogen inactivation in blood banking, stem cell transplantation in leukemia as well as nonmalignant diseases, the reparative potential of stem cells, a new oral antithrombotic therapy and a new class of highly selective factor Xa inhibitors.

Dipyridamole induces changes in the thrombogenic properties of extracellular matrix generated by endothelial cells in culture.

Dipyridamole (DIP) is a drug widely used as an antiplatelet agent, which also has effects on endothelial cells. In this study, the effects of treating confluent endothelial cell monolayers (EC) with DIP on EC viability (trypan blue exclusion test) and metabolic activity (3H-thymidine incorporation) were examined. Platelet reactivity of the extracellular matrix (ECM) produced by untreated and DIP-treated ECs was determined morphometrically by a perfusion technique. Levels of ECM-associated von Willebrand factor (vWF) and fibronectin (FN) were also quantified (ELISA). The present results indicate that treatment of EC with 10 microM DIP did not reduce EC viability but that the incorporation of labelled nucleotides was significantly decreased (p less than 0.01). Platelet deposition onto the ECM generated by DIP-treated cells, perfused at a shear rate of 1300 sec-1, differed significantly with respect to controls (p less than 0.05), and platelet adhesion was also reduced (25% less, p less than 0.05). This effect was shear rate dependent, as no differences were noted when the ECMs were perfused at 300 sec-1 shear rate. Levels of VWF and FN associated with ECM remained unchanged with respect to controls. These results suggest that treatment with DIP alters EC metabolic activity, which in turn, influences the reactivity of the ECM generated by treated cells.

The role of platelets in cancer metastasis.

Clinical, experimental and ultrastructural studies strongly suggest a role for platelets in metastatic dissemination. Several mechanisms have been proposed to explain the potential contribution of blood platelets to the metastatic cascade. Experimentally, many tumour cells of either animal or human origin have the capacity to activate platelets, although the mechanisms by which malignant cells exert this effect is not yet fully understood. Possible mechanisms include: (1) generation of thrombin; (2) activation by ADP; (3) release of cathepsin B; (4) eicosanoid metabolism. A number of observations also indicated that tumour-cell-induced platelet aggregation required specific receptor sites. We have shown that platelet glycoprotein GPIb and the complex GPIIb/IIIa are necessary for tumour-cell-induced platelet aggregation. We and others reported the isolation of a microparticulate aggregating material from different types of tumour cell lines. This material has been identified as a sialolipoprotein complex which possesses tissue-factor-like activity. The role of sialic acid in the metastatic potential of cells is also believed to be important and may partly modulate their interactions with platelets. In vivo, rheological factors may also regulate the interactions of tumour cells with blood and vascular structures and an alternative approach to the evaluation of platelet-tumour-cell interaction under dynamic conditions has been the use of perfusion systems. Thus, we have established the crucial role of Ca2+ in supporting tumour-cell-platelet activation and subsequent thrombus formation. More recently we investigated the patterns of adhesion of a highly metastatic human adenocarcinoma of the lung to exposed extracellular matrix generated by human vascular endothelial cells in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Redistribution of membrane glycoproteins in platelets activated under flow conditions.

A reduction in the ability of GPIb to bind specific MoAbs or ligands (vWF) has been reported in platelets exposed to thrombin in suspension. We have analyzed modifications in the presence of glycoproteins (GPs) on platelets activated under flow conditions in a system which allows limited thrombin and fibrin generation. Normal blood anticoagulated with low molecular weight heparin (LMWH, Dalteparin 20 IU/ml) was recirculated for up to 10 min at 800 s-1 through annular chambers containing denuded arterial segments. Aliquots of blood were removed from the reservoir at 0, 1, 5 and 10 min and immediately mixed with paraformaldehyde. Membrane glycoproteins: GPIb (CD42b), GPIIb-IIIa (CD41a), GPIV (CD36); and activation dependent antigens: P-selectin (CD62P) and lysosomal glycoprortein (CD63), were detected in whole blood by dual color flow cytometry. Circulation of through the perfusion system resulted in platelet activated as demonstrated by the increased percentage of platelets positive for antigens CD62P and CD63. A gradual increase in the binding of MoAbs directed against GPIb, GPIIb-IIIa, and GPIV epitopes was noted during the entire perfusion period. Observed differences in mean fluorescence intensities at all the observation times were statistically significant (P < 0.001). Our results obtained on platelets in an experimental thrombosis system indicate that GPIb, GPIIb-IIIa and GPIV remain on the surface of activated platelets and actually increase their expression. Alterations detected at the level of GPIb in platelets activated by thrombin in suspension may not take place under in vivo situations.