Platelet-subendothelium Interaction Research Articles

Vonicog alfa for the treatment of von Willebrand disease

ABSTRACTIntroduction: von Willebrand disease (VWD) is the most frequent inherited bleeding disorder. It is caused by the quantitative and/or qualitative abnormalities of von Willebrand factor (VWF), an adhesive protein crucial for platelet-subendothelium interaction and for factor VIII stabilization in plasma. Clinical manifestations are mainly represented by excessive mucocutaneous bleeding and prolonged oozing after surgical procedures. The aim of therapy in VWD is to correct the abnormal platelet adhesion-aggregation as a result of low or dysfunctional VWF and the abnormal intrinsic coagulation owing to low FVIII levels. Current therapeutic options are desmopressin, which releases endogenous VWF from endothelial cells, and exogenous VWF contained in plasma-derived VWF/FVIII concentrates.Areas covered: This paper focuses on the potential therapeutic benefits for VWD of the first recombinant VWF concentrate, vonicog alfa. Topics covered include clinical development, pharmacokinetic profile, safety and efficacy data from Phase 1 and 3 clinical studies of vonicog alpha in patients with clinically severe VWD.Expert opinion: Vonicog alfa may provide advantages in treatment of VWD because it is a recombinant high-purity VWF, with a high efficacy profile when co-administered with FVIII as well as alone. Due to its efficacy even without FVIII, vonicog alfa offers the possibility of a more targeted, personalized replacement therapy based on the needs of each individual patient.

Effects of Bemiparin, Dalteparin, and Unfractionated Heparin on Platelet Interaction With Human Subendothelium Under Flow Conditions

We compared the effects of the low-molecular-weight heparin (LMWH), bemiparin, and dalteparin with unfractionated heparin (UFH) on the platelet subendothelium interaction under flow conditions. All three compounds decreased the percentage of subendothelial matrix covered by platelets, although the effect of LMWH was greater than that of UFH. Subendothelial matrix covered with platelet structures greater than 100 square microns was significantly reduced by all three compounds; the effect of bemiparin tended to be greater than that of the other two heparins.

In vitro effects of clopidogrel on the platelet-subendothelium interaction, platelet thromboxane and endothelial prostacyclin production, and nitric oxide synthesis.

Clopidogrel is an antiplatelet drug that belongs to the group of thienopyridines. Because of its main mechanism of action most studies of clopidogrel have centered on the platelet ADP pathway. The aim of the present study was to compare the effects of clopidogrel, ticlopidine, and aspirin, on platelet activation by collagen (the main inducer of platelet activation in vivo), prostanoid, and NO production, and the effects on blood perfusion experiments. Clopidogrel inhibited platelet aggregation induced in whole blood by collagen and TxB2 production to a greater extent than did ticlopidine. Prostacyclin synthesis did not change after incubation with thienopyridines, whereas aspirin inhibited synthesis in a dose-dependent manner. Thienopyridines increased NO production to a greater extent than did aspirin. All three drugs impaired the platelet-subendothelium interaction under flow conditions. With thienopyridines, the presence of endothelium did not modify the percentage of the surface coated by platelets.

Influence of glucose concentration on the effects of aspirin, ticlopidine and clopidogrel on platelet function and platelet–subendothelium interaction

Clinical studies have shown that the ability of aspirin to prevent cerebrovascular accidents is weaker in patients with diabetes. The aim of this study was to determine whether high concentrations of glucose modified the effect of aspirin, ticlopidine and clopigodrel on platelet function and platelet–subendothelium interactions. This in vitro study tested three different concentrations of glucose. The effects were analyzed by comparing platelet aggregometry in whole blood, nitric oxide and prostacyclin production in cultures of human endothelial cells, and by quantitative analysis of morphological features of the platelet–subendothelium interaction under flow conditions. High concentrations of glucose increased platelet aggregation (13.9 Ω with 5 mM glucose vs. 21.6 Ω with 16.6 mM) and platelet–subendothelium interactions (28.9% with 5 mM glucose vs.35.2% with 16.6 mM), and decreased nitric oxide and prostacyclin production. In the presence of high concentrations of glucose, the antiaggregant effect of aspirin and its influence on nitric oxide production were diminished (IC 50 54 μM with 5 mM glucose vs.556 μM with 16.6 mM glucose), and its effect on the platelet–subendothelium interaction was reduced (10.5% platelet occupancy with 5 mM glucose vs.23% with 16.6 mM glucose). The effects of ticlopidine and clopidogrel were not significantly modified.

The discovery of the antiplatelet effect of aspirin: a personal reminiscence

The discovery of the antiplatelet effect of aspirin: a personal reminiscence

Effects of Camonagrel, a Selective Inhibitor of Platelet Thromboxane Synthase, on the Platelet-Subendothelium Interaction

The aim of this study was to compare the effects of a new thromboxane synthase inhibitor, camonagrel, on platelet aggregation and platelet-subendothelium interaction under flow conditions, in comparison with a standard thromboxane synthase inhibitor (dazoxiben) and a cyclooxygenase inhibitor (acetylsalicylic acid). With respect to platelet aggregation in whole blood, the 50% inhibitory concentrations (IC₅₀) of camonagrel were between 318 and 797 µmol/l after induction with collagen and adenosine 5′-diphosphate, respectively. For inhibition of thromboxane B₂ synthesis, the IC₅₀ values were 868 ± 68 µmol/l; prostaglandin E₂ was inhibited only by acetylsalicylic acid (IC₅₀ for camonagrel >2,000 µmol/l), and the leukocyte 6-keto-PGF_1α level was increased by camonagrel. The greatest reduction in percentage subendothelial surface occupied by platelets (mainly in the thrombi) after blood perfusion was seen after incubation with camonagrel in the range of concentrations that inhibited collagen-induced platelet aggregation. In conclusion, camonagrel reduced platelet-subendothelium interaction under flow conditions, showing this effect in a range of concentrations lower than in inhibition of platelet aggregation.

Effects of the selective inhibition of platelet thromboxane synthesis on the platelet-subendothelium interaction.

1. Drugs that inhibit TxA(2) synthesis are used to reduce platelet aggregation. The aim of this study was to compare the effects of a cyclo-oxygenase (COX) inhibitor (acetylsalicylic acid, ASA), a thromboxane synthetase (TxS) inhibitor (dazoxiben) and a dual TxS inhibitor and TxA(2) receptor blocker (DT-TX 30) on platelet aggregation and the platelet-subendothelium interaction in flow conditions. 2. The techniques used in this in vitro study were platelet aggregometry in whole blood, and measurement of platelet thromboxane B(2) and prostaglandin E(2) production and leucocyte production of 6-keto-PGF(1alpha). The platelet-subendothelium interaction was evaluated in rabbit aorta subendothelium preparations exposed to flowing blood at a shear stress of 800 s(-1). Morphometric methods were used to calculate the percentage of subendothelium occupied by platelets. 3. The 50% inhibitory concentration (IC(50)) of DT-TX 30 in whole blood was in the range of 10(-7) micro M (induced with collagen or arachidonic acid) to 10(-5) micro M (induced with thrombin) or 10(-4) (induced with ADP). IC(50) values under all experimental conditions were lower with DT-TX 30 than with ASA. For thromboxane B(2) the IC(50) were: ASA 0.84+/-0.05 micro M, dazoxiben 765+/-54 micro M, DT-TX 30 8.54+/-0.60 micro M. Prostaglandin E(2) was inhibited only by ASA (IC(50) 1.21+/-0.08 micro M). Leucocyte 6-keto-PGF(1alpha) was inhibited by ASA (IC(50) 6.58+/-0.76 micro M) and increased by dazoxiben and DT-TX 30. The greatest reduction in percentage subendothelial surface occupied by platelets after blood perfusion was seen after treatment with DT-TX 30 in the range of concentrations that inhibited collagen-induced platelet aggregation (control group: 31.20+/-3.8%, DT-TX 30 at 0.1 micro M: 10.71+/-0.55%, at 1.0 micro M: 6.53+/-0.44%, at 5.0 micro M; 1.48+/-0.07%). All three drugs reduced thrombus formation, although ASA (unlike dazoxiben or DT-TX 30) increased the percentage surface occupied by adhesions. 4. In conclusion, the effect of specific blockage of TxS together with blockage of membrane receptors for TxA(2) can surpass the effect of ASA in inhibiting the platelet-subendothelium interaction in flow conditions.

A Monoclonal Antibody to Platelet Type III Collagen-binding Protein (TIIICBP) Binds to Blood and Vascular Cells, and Inhibits Platelet Vessel-wall Interactions

TIIICBP is a new platelet receptor involved in platelet-type III collagen and platelet-subendothelium interactions. This receptor is composed of a doublet of 72-68 kDa proteins. In this study, the major protein (68 kDa) was purified and used to produce monoclonal antibodies. One of these antibodies, 7F4, binds to platelets as confirmed by flow cytometry. 7F4 inhibited platelet contact, spreading and aggregation induced by type III collagen. Under flow conditions, 7F4 prevented platelet interactions with type III collagen, endothelial cell matrix and the KOGEOGPK type II collagen octapeptide: the specific sequence recognized by TIIICBP. On the other hand, 7F4 had no effect on platelet-type I collagen interactions. TIIICBP was also detected on lymphocytes, granulocytes and monocytes. TIIICBP was expressed on endothelial cells and fibroblasts but not on smooth-muscle cells. These results show that TIIICBP is expressed on several cell types and participates in cell adhesion to the subendothelium.

Abnormal platelet cytoskeletal assembly in hemodialyzed patients results in deficient tyrosine phosphorylation signaling

Uremic patients have a bleeding tendency associated with a platelet dysfunction. We evaluated the impact of a repeated hemodialysis procedure on primary hemostasis by analyzing different aspects of platelet activation in uremic patients. Studies were performed in (1) eight patients with end-stage renal disease before the hemodialysis program was initiated and after initiating hemodialysis treatment, and in (2) eight patients on maintenance hemodialysis who were transferred to continuous ambulatory peritoneal dialysis. Studies included routine platelet aggregations and evaluation of platelet-subendothelium interactions under flow conditions. Contractile proteins and tyrosine phosphorylation associated with the cytoskeleton were analyzed, before and after thrombin activation of platelets, by electrophoresis after Triton X-100 extraction. No changes in the clinical parameters analyzed were observed among the different study groups. Aggregation and platelet adhesion only improved when patients were shifted from hemodialysis to continuous ambulatory peritoneal dialysis (P < 0.05 for both percentage of surface covered by platelets and aggregate formation). The association of cytoskeletal proteins in platelets from patients under hemodialysis treatment was statistically decreased with respect to the corresponding values in platelets from patients not subjected to dialysis (P < 0.01 for actin). However, after two months on peritoneal dialysis, these values increased to almost control values (P < 0.001 for actin, vs. hemodialysis). Similarly, translocation of tyrosine-phosphorylated proteins to the cytoskeletal fraction was impaired in platelets from hemodialyzed patients, and it recovered partially after the patients transferred to continuous ambulatory peritoneal dialysis. Our present data support the concept that repeated platelet stress during hemodialysis has a deleterious effect on the organization of platelet cytoskeleton, which seems to impair the translocation of signal transduction proteins within platelets compromising the platelet function in uremia.

Platelet concentrates promote procoagulant activity: evidence from experimental studies using a perfusion technique.

Evaluation of the hemostatic effectiveness of platelet transfusions is difficult. Perfusion methods have been employed to test the quality and function of platelet concentrates, allowing differentiation between platelet-platelet and platelet-surface interactions. A study was performed to investigate platelet adhesive and cohesive properties as well as the formation of fibrin when aliquots of platelet concentrates were added to thrombocytopenic blood. Blood previously anticoagulated with low-molecular-weight heparin (20 U/mL) underwent platelet and white cell reduction by filtration. Perfusates were prepared by adding to filtered blood platelets obtained from standard concentrates (stored for 1, 3, and 5 days). The final platelet count in these perfusates was standardized at 80,000 per microL. After perfusions, platelet-subendothelium interaction and fibrin formation were analyzed morphometrically. Results were always compared with those obtained in unfiltered blood (> 150,000 platelets/microL). A slight impairment in the ability of stored platelets to interact with the subendothelium was noticed during the storage period. However, the presence of fibrin was significantly greater than that observed in studies with unfiltered blood (Day 1 = 23.48 +/- 9.43%*; Day 3 = 26.99 +/- 6.74%*; Day 5 = 17.95 +/- 9.06% vs. unfiltered blood = 12.60 +/- 3.08%; *p < 0.05). The lower platelet counts (80,000/microL) in the perfusates containing platelets from concentrates could account for the reduced platelet-subendothelium interactions, but they cannot explain the increments in fibrin formation. While the preparation and storage of platelets have a detrimental effect on platelet adhesiveness, such procedures can positively influence the platelet procoagulant activity necessary to platelet hemostasis.

Effects of triflusal and its main metabolite HTB on platelet interaction with subendothelium in healthy volunteers.

The ex vivo effect of triflusal and acetylsalicylic acid (ASA) on platelet interaction with the subendothelium using the Baumgartner perfusion system (wall shear rate 350 s-1) was assessed in blood from 10 healthy volunteers who given a 15-day course of triflusal 600 mg per day and ASA 400 mg per day in a cross-over trial. The percentage of platelets on the subendothelium showed a decrease of 62% in samples from subjects on ASA and a decrease of 93% in those from subjects on triflusal (P < 0.005). The percentage of the subendothelial surface covered by platelets was reduced by 23.3% after treatment with ASA, mainly due to inhibition of aggregates (75.2%), and by 29.9% after treatment with triflusal, mainly due to inhibition of aggregates (89.6%) and of adhesion (25%). The subendothelial surface covered by activated platelets (adhesions and thrombi) showed 32.5% inhibition after treatment with triflusal and 11.6% after treatment with ASA (P < 0.043 vs. triflusal). In the in vitro experiments, 10 mumol.l-1 triflusal did not modify the percentage of the subendothelium covered by platelets. HTB 1 mmol.l-1 inhibited adhesion (26%) and aggregates (18%). We conclude that HTB participates in the ex vivo effects of triflusal on the platelet-subendothelium interaction.

Effect of Plasma from Patients with Primary Antiphospholipid Syndrome on Platelet Function in a Collagen Rich Perfusion System

The effect on platelet function of plasma from 9 patients with primary antiphospholipid syndrome (PAS) with previous thrombotic episodes was investigated under flow conditions. Five asymptomatic individuals with antiphospholipid antibodies (aPL) (A-aPL) and 14 normal controls were also studied. Patients and controls plasmas were added (1:20 v/v) to anticoagulated blood and perfused through annular chambers containing collagen rich vessel segments. The interaction of platelets with vessel subendothelium was morphometrically evaluated in thin sections. An increase in both covered surface and thrombi formation was observed in perfusions in the presence of PAS-plasma (mean +/- SD: 34.2% +/- 9.6% and 23.2% +/- 10.0% respectively) compared with control plasmas (21.4% +/- 7.3% and 10.1% +/- 7.7%, p < 0.01). Affinity purified anticardiolipin antibodies from one PAS patient showed a similar effect when added to normal blood. In contrast, A-aPL plasma had no effect on platelet-subendothelium interaction. In parallel studies, the same plasmas were incubated with isolated normal platelets before and after activation with ADP or collagen and the binding of immunoglobulins (Ig) was determined by flow cytometry. A significantly increased binding of Ig was observed in 8 out of 9 plasmas from PAS patients when platelets had been activated with collagen but not when resting or ADP activated platelets were used. No increased Ig binding to platelets was seen using A-aPL individuals plasma. These observations might help to explain the pathophysiology of the thrombotic events occurring in patients with PAS.

Platelet Aggregation on Extracellular Matrix: Effect of a Recombinant GPIb-Binding Fragment of von Willebrand Factor

Platelets in whole blood incubated on extracellular matrix (ECM) produced by bovine corneal endothelial cells under oscillatory flow conditions demonstrate extensive aggregate formation. Since both platelet-subendothelium and platelet-platelet interactions are mediated by von Willebrand factor (vWF), we used this system to examine the effect of a recombinant GPIb-binding fragment of vWF (designated RG12986), comprising residues 445-733 of the native vWF subunit, on platelet reactivity with ECM. The seven cysteines present in the RG12986 fragment were reduced and alkylated in order to achieve a monomeric conformation. The recombinant vWF fragment binds to unstimulated platelets in the absence of exogenous modulators. When added to platelet-rich plasma, it inhibits ristocetin-induced platelet agglutination. Binding of 51Cr-labeled platelets in reconstituted whole blood to ECM was inhibited by RG12986 in a dose dependent and saturable manner, with IC50 of 4 microM and maximal inhibition (about 70%) at 6 microM. Scanning electron microscope (SEM) analysis showed that addition of RG12986 to whole blood significantly inhibited platelet aggregation on ECM. The extent of inhibition observed with RG12986 at a final concentration of 4 microM was similar to that obtained with the cell adhesion peptide RGDS at the concentration of 0.1 mM. The ability of the RG12986 fragment to inhibit platelet aggregation on ECM is in agreement with the concept that blockade of vWF-GPIb interaction may inhibit further events leading to activation of the glycoprotein IIb/IIIa (GPIIb/IIIa) complex and subsequent thrombus formation.

Evaluation of the transfusion effectiveness of various platelet concentrates by means of an in vitro perfusion technique

An in vitro perfusion technique was used for the assessment of the effectiveness of platelet concentrates (PCs) administered to patients with bone marrow failure. Denuded rabbit arterial segments mounted in annular chambers were exposed to nonanticoagulated blood drawn directly from the antecubital vein. This perfusion system allows the direct visualization of platelet-subendothelium interactions. The deposition of platelets on exposed subendothelium and the presence of fibrin were evaluated morphometrically. Perfusions were performed before and 10 and 120 minutes after the transfusion of PCs that had been obtained by apheresis and administered immediately after collection (A) or separated by centrifugation of blood units and transfused 24 hours after storage at 22 degrees C (B) or 4 degrees C (C). Bleeding times were also determined systematically. The deposition of platelets on the subendothelium increased significantly (p less than 0.01) 10 minutes after transfusions of A and B PCs, but the presence of fibrin did not differ from pretransfusion values. Two hours after the administration of A or B PCs, the improvement of platelet deposition persisted. By then, the presence of fibrin increased markedly (p less than 0.01). The deposition of platelets was not modified after the transfusion of C PCs, but the presence of fibrin was augmented significantly at both posttransfusion times. Comparisons between bleeding times and morphometric results pointed to a possible role of fibrin formation in contributing to primary hemostasis.

Platelet Adhesion to Subendothelial Structures under Flow Conditions: No Effect of the Lipoxygenase Product 13-HODE

It has been shown that endothelial cells can convert linoleic acid to 13-hydroxyoctadecadienoic acid (13-HODE) and it has been suggested that 13-HODE has non-thrombogenic properties. However, no direct evidence has been presented that indicates that 13-HODE indeed modulates platelet-vessel wall interaction. In this study we have bound a purified 13-HODE to a thrombogenic surface and its effect on platelet adhesion was studied and compared to the effects of an analogous hydroxy fatty acid, 15-hydroxyeicosatetraenoic acid (15-HETE). The effect of 13-HODE on platelet adhesion was studied both under static and flow conditions. In this report we show that binding of up to 40 times the physiological concentration to a thrombogenic surface has no inhibitory effect on platelet adhesion under static or flow conditions. We conclude that 13-HODE is not an important regulatory substance in platelet-subendothelium interaction, although this does not exclude it has a putative anti-adhesive role on intact endothelium.

Sex-related differences in the effects of aspirin on the interaction of platelets with subendothelium.

Using the Baumgartner perfusion technique, marked sex-related differences in the extent of platelet-subendothelium interaction and in the effect of aspirin (ASA) have been observed. The administration of ASA (150 mg daily for 15 days) to two groups of healthy volunteers, one composed of males and the other of females, proved to block the generation of TXB2 in both cases. The basic pattern of platelet subendothelium interaction, however, was found to be markedly different in both groups studied. In men, aspirin treatment induced a significant reduction in the percentage of platelet thrombi, whereas in women, post ASA values remained at the same level as in control experiments. These results show that in the Baumgartner perfusion system women display a less thrombogenic tendency than men and that 150 mg of ASA administered daily are effective in reducing the extent of platelet-subendothelium interaction in the male group but not in the female group. These findings could explain the absence of benefit observed for women in clinical trials with aspirin.

PROLONGED BLEEDING TIME IN CHILDREN AND YOUNG ADULTS WITH HEMOPHILIA

Of 49 patients with hemophilia A or B, who had not received replacement transfusions for at least 72 hours, ten were demonstrated to have a prolonged value for the bleeding time. None had evidence of von Willebrand's disease or prior aspirin ingestion, and only one was found to have an associated intrinsic platelet defect. It appears that the bleeding time, generally thought to be a measurement of platelet-subendothelium interaction, may also sometimes be prolonged in congenital coagulation disorders in which platelet function is normal.

Prolonged Bleeding Time in Children and Young Adults with Hemophilia

Of 49 patients with hemophilia A or B, who had not received replacement transfusions for at least 72 hours, ten were demonstrated to have a prolonged value for the bleeding time. None had evidence of von Willebrand's disease or prior aspirin ingestion, and only one was found to have an associated intrinsic platelet defect. It appears that the bleeding time, generally thought to be a measurement of platelet-subendothelium interaction, may also sometimes be prolonged in congenital coagulation disorders in which platelet function is normal.

Adhesion and aggregation of human platelets to rabbit subendothelium. A new approach for investigation: specific antibodies.

An SgG antibody occurring in a recently transfused thrombasthenic patient inhibited all the ADP-mediated aggregations and platelet-platelet interaction (thrombus formation) on rabbit aorta subendothelium; another IgG antibody occurring in a multitransfused Bernard-Soulier patient inhibited ristocetin and bovine factor VIII mediated aggregation and platelet-subendothelium interaction.