Initial Platelet Contact Research Articles

Shear-dependent suppression of platelet thrombus formation by phosphodiesterase 3 inhibition requires low levels of concomitant Gs-coupled receptor stimulation

Phosphodiesterase (PDE)3 inhibitors exert potent antiplatelet effects through maintaining elevated intracellular cyclic adenosine monophosphate levels, but do not prolong bleeding time. To resolve this discrepancy, we hypothesised that PDE3 inhibitors effectively suppress shear-induced platelet thrombus formation initiated by the interaction of the platelet receptor GPIb/V/IX with its ligand, von Willebrand factor (VWF), since arterial thrombosis is more dependent on shear stress as compared with haemostatic plug formation. To test the hypothesis, we compared the in vitro effects of K-134 (a PDE3 inhibitor), tirofiban (a GPIIb/IIIa inhibitor) and acetylsalicylic acid (ASA) on ristocetin-induced platelet aggregation and platelet thrombus formation on VWF or collagen surfaces under flow conditions. K-134 inhibited GPIIb/IIIa-dependent platelet aggregation to the same extent as tirofiban and more potently than ASA. Likewise, K-134 and tirofiban effectively inhibited stable platelet thrombus formation (platelet firm adhesion and subsequent aggregation) on the VWF or collagen surface under high shear, but ASA only inhibited aggregation. Notably, inhibition by K-134 became evident only when a low concentration of PGE1 was present. These inhibitors did not block shear-induced initial platelet contact with VWF via GPIb/V/IX. In contrast, under low shear, the inhibitory effects of K-134 on platelet aggregation on the collagen surface were lower than tirofiban or ASA. The observed shear-dependent suppression of platelet thrombus formation by PDE3 inhibitor in the presence of low levels of adenylate cyclase stimulator may contribute to high therapeutic benefit with low risk of bleeding.

Abstract 1656: Vegf Induces vWF Release via A VEGFR2/Tyr1175-PLC- gamma1 Pathway

Many endothelial inflammatory and prothrombotic mediators are stored in and rapidly released from Weibel-Palade bodies (WPBs), endothelium-specific storage organelles upon stimulation. The von Willebrand factor (vWF), a major component inside WPBs, mediates the initial contact of platelets with the injured vessel wall and thus plays an important role in haemostasis and thrombosis. It has previously been shown that vascular endothelial growth factor (VEGF) triggers a rapid release of vWF. However, specific VEGF receptors and their potential downstream pathways involved in this process have not been carefully determined. To dissect the role of VEGF receptors in vWF release activation, we utilized two approaches: one is to use receptor-specific ligands and the other is to use a chimeric receptor approach. The ligands for VEGF receptor 2 (VEGFR2), but not VEGF receptor 1 (VEGFR1), stimulated vWF release. The predominant role of VEGFR2 in vWF release regulation was further confirmed by using a chimeric receptor approach in which the extracellular domain of the epidermal growth factor receptor was substituted for that of VEGFR1 or VEGFR2, and was then expressed in early-passage HUVECs. Further, the knockdown of phospholipase C-γ1 (PLCγ1) suppressed VEGF-triggered intracellular calcium increase and blocked VEGF-induced vWF release. In addition, the two products of PLCγ1 hydrolysis, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), are both required for VEGF-induced vWF release. Finally, combined with point mutagenesis, the responsible binding sites for PLCγ1 and their importance in VEGFR2-activated vWF release have been determined. Point mutation of a single tyrosine residue Tyr1175, a putative binding site for PLCγ1 on the C-terminus, abolished VEGFR2-activated vWF release. This study presents the first evidence that the PLCγ1 is essential for VEGF-triggered vWF release mediated through a VEGFR2/Tyr1175 pathway.

Von Willebrand factor and factor VIII are independently required to form stable occlusive thrombi in injured veins

Abstractvon Willebrand factor (VWF) protects factor VIII (FVIII) from proteolysis and mediates the initial contact of platelets with the injured vessel wall, thus playing an important role in hemostasis and thrombosis. VWF is crucial for the formation of occlusive thrombi at arterial shear rates. However, with only a few conflicting studies published, the role of VWF in venous thrombosis is still unclear. Using gene-targeted mice, we show that in ferric chloride–injured veins platelet adhesion to subendothelium is decreased and thrombus growth is impaired in VWF−/− mice when compared with wild type (WT). We also observed increased embolization in the VWF−/− mice, which was due to lower FVIII levels in these mice as recombinant factor VIII (r-FVIII) restored thrombus stability. Despite normalization of blood clotting time and thrombus stability after r-FVIII infusion, the VWF−/− venules did not occlude. Transgenic platelets lacking the VWF receptor GPIbα extracellular domain showed decreased adhesion to injured veins. But, after a delay, all the injured venules occluded in these transgenic mice. Thus, VWF likely uses other adhesion receptors besides GPIbα in thrombus growth under venous shear conditions. Our studies document crucial roles for VWF and FVIII in experimental thrombosis under venous flow conditions in vivo.

Outside-In Signaling by Integrin αIIbβ3 Is Dramatically Affected by the Density of Adsorbed Fibrinogen and Results in Differences in Calcium Mobilization, Integrin Engagement, and Platelet Morphology.

αIIbβ3-mediated outside-in signaling following platelet adhesion to fibrinogen affects platelet adhesion, degranulation, and spreading, but the precise mechanisms involved remain incompletely understood. In this study, we demonstrate that the character of αIIbβ3 signaling is dependent on the surface density of the adsorbed ligand. Time-lapse total internal reflection fluorescence microscopy (TIR-FM) allowed us to visualize the interactions between fibrinogen and the αIIbβ3 integrins on the basal membrane of adhering platelets using a fluorescently-labeled antibody specific for β3. We observed significant morphological and biochemical differences in the initial interaction between αIIbβ3 and fibrinogen depending on the fibrinogen surface density. These included differences in the kinetics of filopodia and lamellipodia formation. Although filopodia started to form at the same time after initial platelet contact with both low-density (adsorbed from 3 μg/ml) fibrinogen (~ 40–50 s), new filopodia formed for a significantly shorter period of time on low- than on high-density fibrinogen [120 s (n = 79) vs. 235 s (n = 62); medians; p< 0.001]. Lamellipodia started to form in platelets adhering to low-density fibrinogen significantly later than in platelets on high-density fibrinogen (140 s vs. 110 s; p = 0.04). Moreover, once lamellipodia started to appear, formation of new filopodia ceased in platelets adhering to low-density fibrinogen. In contrast, in platelets on high-density fibrinogen new filopodia continued to form even in the presence of lamellipodia for another 155 s. We also observed that a higher percentage of platelets adherent to high-density fibrinogen (52 ± 6 %) failed to develop a cytoplasmic Ca2+ signal compared to platelets adherent to low-density fibrinogen (13 ± 11 %; n = 3; p<0.001). In addition, only 8 ± 6 % of platelets on high-density fibrinogen developed sustained Ca2+ oscillations compared to 39 ± 14 % of platelets on low-density fibrinogen (n = 3; p = 0.01). TIR-FM analysis of platelets stained with a monoclonal antibody specific for a ligand-induced binding site in the PSI domain of β3 (AP5), showed that on low-density fibrinogen only receptors in a very thin and stationary ring at the edge of fully spread platelets were stained, whereas on high-density fibrinogen AP5 stained a thicker, more diffuse, and more mobile ring. These observations suggested that αIIbβ3 interactions with high-density fibrinogen are more transient than those with low-density fibrinogen. To test this directly, platelets adherent to fibrinogen for 1 h were treated with αIIbβ3 antagonists or EDTA and the loss of adhesion was then measured. Treatement with c7E3, tirofiban, or EDTA resulted in loss of 66 ± 13%, 57 ± 21%, and 63 ± 25%, respectively, of platelets on high density fibrinogen. In contrast, the same treatments resulted in loss of only 25 ± 3%, 34 ± 14%, and 31 ± 21% of platelets on low-density fibrinogen (n = 3; p ≤ 0.01 for each treatment). Thus, we provide evidence that the density of a single immobilized ligand can profoundly affect the morphological and biochemical nature of events leading to platelet adhesion, spreading, and outside-in signaling. These observations have potential significance for hemostasis, thrombosis, and the development of biocompatible materials.

Von Willebrand Factor and Factor VIII Are Independently Required To Form Stable Occlusive Thrombi in Injured Veins.

von Willebrand factor (VWF) is a large adhesive glycoprotein synthesized in megakaryocytes and endothelial cells and stored in platelet a-granules and Weibel-Palade bodies respectively. It protects Factor VIII (FVIII) from proteolysis and mediates the initial contact of platelets with the injured vessel wall thus playing an important role in hemostasis and thrombosis. VWF is crucial for the formation of occlusive thrombi at arterial shear rates. However, with only a few conflicting studies published, the role of VWF in venous thrombosis is still unclear. Therefore in order to understand the in vivo role of VWF and FVIII in experimental thrombosis under venous flow conditions, we decided to evaluate thrombosis in VWF−/−, FVIII−/− and transgenic mice lacking the GPIbα extracellular domain which was replaced by human Interleukin-4 receptor (IL4Rα/GPIbα-tg). In ferric chloride-injured veins, platelet adhesion to subendothelium is decreased and thrombus growth is impaired in the VWF−/− mice when compared to wild-type (WT). In the WT mice, thrombi grew to occlusive size with a mean time of 18 min and all injured venules occluded, whereas in VWF−/− mice none of the vessels occluded by 40 min after injury, when observation was terminated. Venules of mice deficient in FVIII treated similarly also did not occlude because of embolization. The infusion of recombinant human-FVIII (r-hu-FVIII) in FVIII−/− mice normalized the occlusion time to WT values. We also observed thrombus instability in the VWF−/− mice, which was due to lower FVIII levels in these mice since r-huFVIII restored thrombus stability i.e. prevented breaking of the thrombi with large platelet aggregates moving downstream. Despite normalization of blood clotting time and thrombus stability after r-FVIII infusion, the VWF−/− thrombi grew at a slower rate than WT and the venules did not occlude. In transgenic mice lacking the GPIbα extracellular domain, all injured venules occluded. Thus, VWF uses other adhesion receptors besides GPIbα in thrombus growth under venous shear conditions. Our studies document crucial independent roles for VWF and FVIII in experimental thrombosis under venous flow conditions in vivo.

Identification of human platelet glycoprotein VI-specific IgG autoantibody and its fragments

Glycoprotein VI (GPVI), as a major receptor for collagen on the platelet surface, mediates the initial platelet contact with collagen, and causes platelet aggregation and thrombosis. Agents of anti-GPVI can inhibit the adhesion, activation and aggregation function of platelets, which can be used for preventing thrombotic diseases. To make humanized monoclonal antibodies by phage surface display technology, we studied plasmas from 44 patients with chronic idiopathic thrombocytopenic purpura using modified monoclonal antibody immobilization of platelet antigen assays. GPVI-specific antibodies were found in six (13.6%) patients. Among these, only one showed significant inhibition of platelet aggregation induced by collagen. The IgG antibody and F(ab')2 fragments of this patient's plasma were further purified and their immunoreactivities and effects on platelet aggregation were reanalyzed. It was found that purified IgG and its F(ab')2 fragments from the patient not only retained the ability to bind to platelet GPVI, but also inhibited collagen-induced platelet aggregation. In this study, therefore, the specific anti-platelet GPVI autoantibody that inhibits collagen-induced platelet aggregation has been successfully screened out, which can be used to develop completely humanized anti-GPVI phage antibody.

Formation of Procoagulant Platelet Derived Microparticles through Platelet GPIb-Interaction with Von Willebrand Factor under High Shear Stress.

We describe here novel findings of the mechanism of initial platelet contact with immobilized von Willebrand factor (VWF) under high shear stress and how this leads to the formation of procoagulant platelet derived microparticles. In a parallel plate perfusion chamber whole blood was perfused over multimeric VWF or dimeric VWF A1 domain at shear rates between 2,000 s−1 and 40,000 s−1. Platelet attachment to VWF always occurred through glycoprotein Ibα receptors located in discrete adhesion points (DAPs), i.e. few limited membrane areas of 0.05 to 0.1 μm2 that arrested the platelets on the surface. The ongoing flow translocated such anchored platelets downstream, thus pulling membrane tethers from the intact and unstimulated platelet. Tethers could remain connected with the platelet body or be eventually severed, which occurred preferentially at shear rates above 6,000 s−1. Depending on the length of the severed membrane fragment they represented either isolated tethers or microparticles (arrowheads; see Figure below), the latter defined by a diameter of 50 to 100 nanometers. The shear rate threshold for microparticle formation was between 6,000 s−1 and 10,000 s−1. Immuno-fluorescence and immuno-electron microscopy showed glycoprotein Ibα clustered in DAPs of microparticles and tethers, i.e. the contact sites with the surface immobilized VWF. The microparticles also exhibited tissue factors on their surface and showed significant procoagulant activity measured by thrombin generation. We propose that after GPIbα anchoring to VWF in flowing blood passive mechanical pulling of membrane from platelets may generate platelet derived microparticles that can potentially support thrombogenesis. [Display omitted]

Integrin Polymorphisms as Risk Factors for Thrombosis

The study of integrin polymorphisms as risk factors for thrombosis is in its infancy. Like many other purported genetic risk factors, controversies abound, and it will take much work to shift through the information and identify those individuals for whom these genetic variations are true risks for thrombosis. There is a clear need for well-designed, large, prospective, genetic epidemiologic studies. Large clinical trials should be helpful in this regard, and genotyping patients in these studies will be the only way to correlate response to therapy with genetic factors (pharmacogenetics). Hand-in-hand with such clinical investigations, in vitro studies of the functional consequences of these inherited traits should provide a sound rationale for future interventions that will ultimately be beneficial to patients.

Mechanisms-Regulated Platelet Spreading after Initial Platelet Contact with Collagen

In static condition, 6F1, an anti-glycoprotein Ia/IIa monoclonal antibody, almost completely prevented initial platelet adhesion to fibrillar collagen, but markedly lost its action with prolonged incubation. The platelet adhesion and spreading at the later stage were prevented by adding the peptide GRGDS, aspirin, and apyrase, suggesting that after initial recognition of platelet glycoprotein Ia/IIa with collagen the activation of glycoprotein IIb/IIIa and the release of thromboxane A2/ADP would promote platelet spreading, thus strengthening the stability of adhesion. Both initial platelet adhesion and platelet spreading were prevented by cytochalasin B, an inhibitor of actin polymerization. In contrast, BAPTA (an intracellular Ca2+chelator) only inhibited platelet spreading. Inhibition of protein kinase C or protein tyrosine kinase by staurosporine or genistein, respectively, had only little effect on platelet adhesion. These data suggest that actin polymerization and intracellular Ca2+mobilization are involved in the regulation of platelet spreading after initial platelet contact with collagen.

9] Nitric oxide in platelets

Publisher Summary This chapter discusses the regulation of nitric oxide (NO) in platelets. Platelets are blood elements that are involved in physiological reactions ensuring the integrity of vessel walls, known as hemostasis. The regulation of platelet function is of paramount importance for vascular homeostasis. Platelet reactions that occur in response to physiological or pathological stimuli are qualitatively similar and form a chain of interdependent and superimposed steps. They are often referred to as adhesion, which denotes an initial platelet contact to altered biological or nonbiological surfaces, and aggregation, which describes dynamic platelet–platelet interactions leading to the formation of a hemostatic plug. The hemostatic plug is then reinforced with fibrin and subsequent fibrinolytic and tissue repair reactions lead to the restoration of normal blood flow. The appreciation of the platelet-regulatory actions of NO stimulates research efforts aim at elucidation of the biological significance of the mediator in vascular homeostasis and hemostasis. One can investigate the hemostatic actions of NO on platelets or use platelets as a sensitive bioassay system to detect NO generated by other biological or nonbiological systems.

Von Willebrand Factor: Structure and Function

Von Willebrand factor (vWF) is an adhesive, multimeric glycoprotein present in plasma, platelets, and subendothelium, which has two main functions: (1) it serves as a carrier for factor VIII and (2) it plays a crucial role in platelet adhesion to subendothelium, acting as a "bridge" between platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa and subendothelial components such as collagen and heparin. vWF is involved at high shear rates in the initial contact of platelets with the subendothelium, in their subsequent spreading, and in thrombus formation. The three pools of vWF (plasma, platelets, and subendothelium) are necessary for optimal adhesion. Specific fragments of vWF involved in binding to platelets, collagen, heparin, and factor VIII have been mapped by using a series of proteases and well-characterized monoclonal antibodies to distinct epitopes of vWF. Several groups, including ours, have identified at least eight functional domains on the 270-kd subunit that consists of 2,050 amino acids. The importance of the binding domains to GP Ib and to collagen is illustrated by the role of vWF fragment SpIII (amino acids 1 through 1,365) in promoting platelet adhesion to collagen. The role of the vWF-GP Ib axis and of the vWF-GP IIb/IIIa axis in platelet-vessel wall interactions has been demonstrated through the study of patients, monoclonal antibodies, recombinant fragments, and synthetic peptides. We have recently expressed fragments of vWF complementary DNA in Escherichia coli. One of these recombinant fragments, which spans amino acids 449 through 730, binds to platelets in the presence of ristocetin, to collagen, and to heparin and has the property of inhibiting ristocetin-induced platelet agglutination. The second fragment, which spans amino acids 914 through 1,364, binds to collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparative study of prostacyclin infusion given before and during cardiopulmonary bypass to assess the first pass effect of the circuit on platelet number and function

Platelet damage during cardiopulmonary bypass (CPB), although proportional to the duration of bypass, may result in significant dysfunction after the initial contact with an extracorporeal circuit, the so-called 'first pass' phenomenon. The platelet sparing effect of prostacyclin (PGI2) infusion was studied in a double-blind randomized trial on male patients undergoing coronary artery bypass grafts to assess the effect of the 'first pass' through the CPB circuit. Prostacyclin infusion was begun before the onset of CPB or during CPB in two groups which were compared to a placebo control group. A standardized anaesthetic, surgical and perfusion technique were used. Preoperatively and during surgery at pre-set intervals, whole blood platelet aggregation was studied using ADP and collagen agonists. Platelet numbers and function measured by ADP aggregation were conserved in the two PGI2 groups. There was no significant difference between the treated groups. We conclude, therefore, that the initial contact of platelets with the CPB circuit, in the absence of PGI2 did not irreversibly affect platelet function. In addition, the hypotensive action of PGI2 was easier to control once on bypass. It may therefore be preferable to delay PGI2 infusion until CPB has been established.

Platelet interaction with subendothelium in flowing rabbit blood: Effect of blood shear rate

An annular perfusion chamber was utilized to expose subendothelium from everted rabbit aorta to blood flowing under controlled conditions. Initial platelet contact ( C), platelet spreading ( S), and the formation of platelet microthrombi ( T) were evaluated by a morphometric technique for exposure times ranging from 1 to 40 min and flow rates of 10, 20, 40, and 160 ml/min (comparable to vessel wall shear rates of 650, 1300, 2600, and 10,000 sec −1). Analysis of the platelet adhesion results ( C + S) with available mass transport theory indicated that, under these shear conditions, both platelet transport to the vicinity of the subendothelium and platelet reactivity with the vessel wall influenced the platelet attachment rate. At the highest shear rate studied (10,000 sec −1) an inhibition of platelet adhesion was observed which may be due to the effect of shear on either platelet transport or reactivity. The rate of formation of platelet microthrombi increased sharply between shear rates of 650 and 1300 sec −1 and continued to increase up to 10,000 sec −1 even though platelet adhesion was inhibited at this shear rate. Below 1000 sec −1 thrombi were always transiently formed, disappearing after exposure times of 20 min. Above 1000 sec −1 platelet thrombi formed from the blood of certain experimental animals had not disappeared at exposures as long as 40 min. The mechanisms governing the inhibition of platelet adhesion and the stabilization of platelet thrombi by shear rates encountered in certain areas of the microcirculation are currently unknown.