Recruitment Of Additional Platelets Research Articles

Colchicine reduces platelet aggregation by modulating cytoskeleton rearrangement via inhibition of cofilin and LIM domain kinase 1

IntroductionPlatelets activation/aggregation with subsequent thrombus formation is the main event in the pathophysiology of acute coronary syndrome. Once activated, platelets show an extensive cytoskeleton rearrangement that leads to recruitment of additional platelets to finally cause haemostatic plug formation. Thus, the cytoskeleton plays a pivotal role in this phenomenon. Colchicine (COLC) is an anti-inflammatory drug proven to reduce major cardiovascular events in patients with coronary artery disease. The molecular mechanisms by which COLC exerts these protective effects remain partially still unknown. Since COLC causes disruption of tubulin, a component of cell cytoskeleton, we investigated whether this drug might interfere with platelet aggregation by acting on cytoskeleton rearrangement. Methods and resultsPlatelets isolated from healthy volunteers were activated with Adenosine Diphosphate (ADP, 20 μM) Collagen (COLL, 60 μg/ml) and Thrombin Activating Receptor Peptide (TRAP 25 μM) with/without COLC 10 μM pretreatment. After stimulus, aggregation was measured by light aggregometry overtime. Microtubules structure was assessed by immunohistochemistry and key proteins involved in regulation of actin-filament assembly and contractility such as Myosin Phosphatase Targeting subunit (MYPT), LIM domain kinase 1(LIMK1) and cofilin were evaluated by Western Blot analysis. Colchicine pretreatment significantly blunted ADP/COLL/TRAP-induced platelet aggregation (up to 40%). COLC effects appeared mediated by microtubules depolymerization and cytoskeleton disarrangement associated to inactivation of MYPT and LIMK1 that finally interfered with cofilin activity. ConclusionsOur data indicate that colchicine exerts anti-platelet effects in vitro via inhibition of key proteins involved in cytoskeleton rearrangement, suggesting that its beneficial cardiovascular properties may be due, at least in part, to an inhibitory effect of platelet activity.

RhoG Protein Regulates Platelet Granule Secretion and Thrombus Formation in Mice

Rho GTPases such as Rac, RhoA, and Cdc42 are vital for normal platelet function, but the role of RhoG in platelets has not been studied. In other cells, RhoG orchestrates processes integral to platelet function, including actin cytoskeletal rearrangement and membrane trafficking. We therefore hypothesized that RhoG would play a critical role in platelets. Here, we show that RhoG is expressed in human and mouse platelets and is activated by both collagen-related peptide (CRP) and thrombin stimulation. We used RhoG(-/-) mice to study the function of RhoG in platelets. Integrin activation and aggregation were reduced in RhoG(-/-) platelets stimulated by CRP, but responses to thrombin were normal. The central defect in RhoG(-/-) platelets was reduced secretion from α-granules, dense granules, and lysosomes following CRP stimulation. The integrin activation and aggregation defects could be rescued by ADP co-stimulation, indicating that they are a consequence of diminished dense granule secretion. Defective dense granule secretion in RhoG(-/-) platelets limited recruitment of additional platelets to growing thrombi in flowing blood in vitro and translated into reduced thrombus formation in vivo. Interestingly, tail bleeding times were normal in RhoG(-/-) mice, suggesting that the functions of RhoG in platelets are particularly relevant to thrombotic disorders.

Inherited platelet disorders and oral health

Platelets play a key role in thrombosis and hemostasis. Accumulation of platelets at the site of vascular injury is the first step in the formation of hemostatic plugs, which play a pivotal role in preventing blood loss after injury. Platelet adhesion at sites of injury results in spreading, secretion, recruitment of additional platelets, and formation of platelet aggregates. Inherited platelet disorders are rare causes of bleeding syndromes, ranging from mild bruising to severe hemorrhage. The defects can reflect deficiency or dysfunction of platelet surface glycoproteins, granule contents, cytoskeletal proteins, platelet pro-coagulant function, and signaling pathways. For instance, Bernard-Soulier syndrome and Glanzmann thrombasthenia are attributed to deficiencies of glycoprotein Ib/IX/V and GPIIb/IIIa, respectively, and are rare but severe platelet disorders. Inherited defects that impair platelet secretion and/or signal transduction are among the most common forms of mild platelet disorders and include gray platelet syndrome, Hermansky-Pudlak syndrome, and Chediak-Higashi syndrome. When necessary, desmopressin, antifibrinolytic agents, and transfusion of platelets remain the most common treatment of inherited platelet disorders. Alternative therapies such as recombinant activated factor VII are also available for a limited number of situations. In this review, we will discuss the management of patients with inherited platelet disorders in various clinical situations related to dental cares, including surgical intervention.

The role of inositol polyphosphate 4-phosphatase 1 in platelet function using a weeble mouse model

Platelet activation plays an important role in the development and course of cardiovascular disease. It is triggered by the interaction of subendothelial matrix-bound and/or soluble agonists with platelet surface receptors causing a series of morphological and biochemical changes leading to the recruitment of additional platelets and formation of stable platelet aggregates. In addition to events causing initial activation and recruitment of platelets, signaling continues post-aggregation that promotes stability of the thrombus (Brass et al., 2004). Platelet levels of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) increase dramatically in response to agonist stimulation in an aggregation-dependent manner (Kucera et al., 1990; Nolan et al., 1990; Sultan et al., 1990). Furthermore the increase in platelet PtdIns(3,4)P2 occurs late in platelet aggregation and correlates with the irreversible phase of platelet aggregation (Sorisky et al., 1992; Sultan et al., 1991; Trumel et al., 1999), suggesting that PtdIns(3,4)P2 mediates the stabilization of platelet aggregates. However little is known about the regulation of PtdIns(3,4)P2 in platelets. PtdIns(3,4)P2 can be formed by three different routes 1) by direct phosphorylation of PtdIns(4)P by phosphatidylinositol 3-kinase (EC2.7.1.153) (PI 3-K) 2) by PI 3-K using PtdIns(4,5)P2 as a substrate followed by dephosphorylation by a 5-phosphatase (EC3.1.3.56) and, 3) by the action of type I PtdIns(4)P phosphate kinase (EC2.7.1.68) using PI 3-K as substrate (Zhang et al., 1998), shown diagramatically in figure 1. It is well documented that different PI 3-K isoforms play important roles in both early and later stages of platelet aggregation (Jackson et al., 2006). It has been shown that that pharmacologic inhibition of PI 3-K prevents agonist-induced formation of PtdIns(3,4)P2 (Kovacsovics et al., 1995; Schoenwaelder et al., 2007). Furthermore addition of PI 3-K inhibitors after the onset of platelet aggregation induces a decline in PtdIns(3,4)P2 and disaggregation of platelets, supporting a role for PtdIns(3,4)P2 in the stabilization of aggregates. However, platelet agonist-dependent activation of PI 3-K mediates an increase in both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 making approaches using pharmacologic inhibition or genetic disruption of PI 3-K unsuitable to distinguish the contributions of the individual 3-phosphorylated phosphoinositides to platelet signaling. The levels of inositol lipids in cells and their distribution in discrete cellular compartments are regulated by the balance of the enzymatic activities of kinases and phosphatases. Figure 1 Pathways for the formation of PtdIns (3,4)P2. The major route of PtdIns(3,4)P2 hydrolysis is the removal of the D4 phosphate by the enzymes cloned and characterized in our lab (Norris et al., 1997; Norris et al., 1995), inositol polyphosphate 4-phosphatase type I (EC3.1.3.40) and type II (EC3.1.3.66) that are magnesium-independent phosphatases. Over their entire sequence type I and type II 4-phosphatases are 37% identical (Norris et al., 1997). The active site region is more highly conserved, and contains a consensus sequence found in other magnesium-independent phosphatases (Zhang et al., 1994). The consensus sequence CX5 RT/S, is conserved throughout 4-ptases from C. elegans to humans as shown in figure 2. These enzymes do not catalyze the hydrolysis of lipids other than PtdIns(3,4)P2 and therefore provide unique means for the study of this lipid in platelet activation. We have shown that 4-ptase I forms a complex with PI 3-K in platelets which localizes the complex to sites of PtdIns(3,4)P2 production (Munday et al., 1999). Figure 2 Alignment of active sites of 4ptases. We postulate that PtdIns(3,4)P2 is important for platelet function and will study this using a mouse model. We previously showed that an antibody that reacts with 4-phosphatases immunoprecipitates all of the PtdIns(3,4)P2 hydrolyzing activity from human platelets (Munday et al., 1999). An early indication that PtdIns(3,4)P2 was important for platelet function was the work of Norris (Norris et al., 1997). It was shown that calpain caused degradation of recombinant 4-phosphatase I in vitro thereby inactivating it. It was also shown that activation of human platelets with either calcium ionophore or thrombin led to proteolysis of endogenous platelet 4-phosphatase I. If calpeptin, a cell-permeable inhibitor of calpain, was included in these experiments no proteolysis was seen. The levels of PtdIns(3,4)P2 in platelets were lower when calpeptin was included, indicating that 4-phosphatase I was important for controlling the levels of PtdIns(3,4)P2 during platelet activation. A naturally occurring mutation in type I 4-phosphatase is a single nucleotide deletion which is found in the weeble mouse. These animals suffer from severe neurodegeneration and die within the first weeks of life. Therefore such mutant mice cannot be used to study platelet function. We circumvented this problem by creating chimeric mice by bone marrow transplantation of weeble fetal liver cells into lethally irradiated wild type mice. These mice lack 4-phosphatase in bone marrow derived cells including platelets. The mice are viable, but lack platelet 4-phosphatase I.

Inherited disorders of platelet function and challenges to diagnosis of mucocutaneous bleeding

Platelets play a pivotal role in the arrest of bleeding at sites of vascular injury. Following endothelial damage, they respond rapidly by adhesion to subendothelial matrix proteins resulting in platelet activation, spreading, aggregation, secretion and recruitment of additional platelets to form the primary haemostatic plug. This mass provides a surface for thrombin generation and fibrin mesh formation that stabilizes the clot. Careful study of patients with inherited platelet disorders and, subsequently, of informative animal models, has identified structural platelet abnormalities that have enhanced our understanding of platelet function. The investigations of rare, but severe, inherited platelet disorders have led us to the discovery of causative molecular defects. One of the most informative is the rare autosomal recessive disorder Glanzmann thrombasthenia, caused by defect or deficiency in the platelet integrin alphaIIbbeta3, resulting in absent platelet aggregation and a significant clinical bleeding diathesis. Our new challenge is to understand the mechanisms underlying more common, but less well-defined, mucocutaneous bleeding (MCB) disorders. Present diagnostic testing for platelet function disorders and von Willebrand's Disease often fails to identify the cause of bleeding in individuals with inherited MCB.

Antiplatelet drugs

SummaryPlatelets play a major role in thromboembolic diseases, and so antiplatelet therapy remains crucial in treatment and prophylaxis. Upon vascular injury, platelets rapidly adhere to the exposed subendothelial matrix, after which they become activated, resulting in the recruitment of additional platelets from the circulation to eventually form a stable arterial platelet plug. Although controlled plug formation is desired for the prevention of excessive blood loss and for promoting wound healing, several pathological conditions may result in the formation of occlusive thrombi leading to severe clinical complications, including myocardial infarction and ischaemic stroke.Many antiplatelet approaches have been investigated, interfering with one or more of the different stages in thrombus formation. This review discusses antiplatelet agents that interfere with the three principal phases in thrombus formation: platelet adhesion, amplification of platelet activation and platelet aggregation. For each stage, novel experimental targets and clinically established antiplatelet strategies will be reviewed. Limitations and possible benefits will be discussed for each target.

Activation of Platelet Function Through G Protein–Coupled Receptors

Because of their ability to become rapidly activated at places of vascular injury, platelets are important players in primary hemostasis as well as in arterial thrombosis. In addition, they are also involved in chronic pathological processes including the atherosclerotic remodeling of the vascular system. Although primary adhesion of platelets to the vessel wall is largely independent of G protein-mediated signaling, the subsequent recruitment of additional platelets into a growing platelet thrombus requires mediators such as ADP, thromboxane A(2), or thrombin, which act through G protein-coupled receptors. Platelet activation via G protein-coupled receptors involves 3 major G protein-mediated signaling pathways that are initiated by the activation of the G proteins G(q), G(13), and G(i). This review summarizes recent progress in understanding the mechanisms underlying platelet activation and thrombus extension via G protein-mediated signaling pathways.

Activation-independent platelet adhesion and aggregation under elevated shear stress

AbstractPlatelet aggregation, which contributes to bleeding arrest and also to thrombovascular disorders, is thought to initiate after signaling-induced activation. We found that this paradigm does not apply under blood flow conditions comparable to those existing in stenotic coronary arteries. Platelets interacting with immobilized von Willebrand factor (VWF) aggregate independently of activation when soluble VWF is present and the shear rate exceeds 10 000 s–1 (shear stress = 400 dyn/cm2). Above this threshold, active A1 domains become exposed in soluble VWF multimers and can bind to glycoprotein Ibα, promoting additional platelet recruitment. Aggregates thus formed are unstable until the shear rate approaches 20 000 s–1 (shear stress = 800 dyn/cm.2). Above this threshold, adherent platelets at the interface of surface-immobilized and membrane-bound VWF are stretched into elongated structures and become the core of aggregates that can persist on the surface for minutes. When isolated dimeric A1 domain is present instead of native VWF multimers, activation-independent platelet aggregation occurs without requiring shear stress above a threshold level, but aggregates never become firmly attached to the surface and progressively disaggregate as shear rate exceeds 6000 s–1. Platelet and VWF modulation by hydrodynamic force is a mechanism for activation-independent aggregation that may support thrombotic arterial occlusion.

Reactive oxygen species: players in the platelet game.

Platelets participate not only in thrombus formation but also in the regulation of vessel tone, the development of atherosclerosis, angiogenesis, and in neointima formation after vessel wall injury. It is not surprising, therefore, that the platelet activation cascade (including receptor-mediated tethering to the endothelium, rolling, firm adhesion, aggregation, and thrombus formation) is tightly regulated. In addition to already well-defined platelet regulatory factors, such as nitric oxide (NO), prostacyclin (PGI2), and adenosine, reactive oxygen species (ROS) participate in the regulation of platelet activation. Although exogenously derived ROS are known to affect the regulation of platelet activation, recent data suggest that the platelets themselves generate ROS. Intracellular ROS signaling in activated platelets could be of significant relevance after transient platelet contact with the vessel wall, during the recruitment of additional platelets, and in thrombus formation. This review discusses the potential cellular and enzymatic sources of ROS in platelets, their molecular mechanisms of action in platelet activation, and summarizes in vitro and in vivo evidence for their physiological and potential therapeutic relevance.

Neuroprotective effect of SolCD39, a novel platelet aggregation inhibitor, on transient middle cerebral artery occlusion in rats.

SolCD39 is a soluble form of recombinant human ecto-ATP/ADPase (NTPDase1) and represents a new class of antithrombotic agents. SolCD39 blocks and reverses platelet activation, preventing recruitment of additional platelets into a growing thrombus. The purpose of this study was to examine the effect of solCD39 on neurological deficit, infarct size, and extent of edema after transient middle cerebral artery occlusion (MCAO) in rats. Physiologically controlled Sprague-Dawley rats underwent 2-hour MCAO by retrograde insertion of an intraluminal suture coated with poly-l-lysine. The agent (solCD39) was administered intravenously before MCAO or at 1-hour or 3-hour recirculation. Other groups received vehicle (Tris-buffered saline) or human albumin (as a "positive" neuroprotective control; 25%, 0.5% of body weight) at 1-hour recirculation. Neurological status was evaluated during occlusion (at 60 minutes) and daily for 3 days after MCAO. Brains were perfusion-fixed at 72 hours, and infarct volumes and brain swelling were determined. Pretreatment with solCD39 significantly improved the neurological score at 72 hours compared with the vehicle group (4.4+/-0.6 versus 7.6+/-0.6, respectively; P=0.008). Cortical infarct areas were significantly reduced at multiple levels by pretreatment with solCD39. Total striatal infarct area was also significantly reduced compared with vehicle by both solCD39 pretreatment (48% mean reduction) and solCD39 treatment at 3-hour recirculation (51% mean reduction). Treatment with SolCD39 significantly reduced total infarct volume (corrected for brain swelling) by an average of 71% to 72% when administered either before ischemia or at 3 hours of recirculation compared with vehicle. Treatment with albumin significantly reduced neurological score and total, cortical, and subcortical infarction at multiple levels, as expected. Treatment with SolCD39, administered either before or at 3 hours after MCAO, improves neurological score and reduces infarct size compared with vehicle. A pharmacological agent of this type appears to have potential for the treatment of focal ischemic stroke.

Sequential cytoplasmic calcium signals in a 2-stage platelet activation process induced by the glycoprotein Ibα mechanoreceptor

We found that the interaction of platelets with immobilized von Willebrand factor (VWF) under flow induces distinct elevations of cytosolic Ca(++) concentration ([Ca(++)](i)) that are associated with sequential stages of integrin alpha(IIb)beta(3) activation. Fluid-dynamic conditions that are compatible with the existence of tensile stress on the bonds between glycoprotein Ibalpha (GPIbalpha) and the VWF A1 domain led to Ca(++) release from intracellular stores (type alpha/beta peaks), which preceded stationary platelet adhesion. Raised levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate, as well as membrane-permeable calcium chelators, inhibited these [Ca(++)](i) oscillations and prevented stable adhesion without affecting the dynamic characteristics of the typical platelet translocation on VWF mediated by GPIbalpha. Once adhesion was established through the integrin alpha(IIb)beta(3), new [Ca(++)](i) oscillations (type gamma) of greater amplitude and duration, and involving a transmembrane ion flux, developed in association with the recruitment of additional platelets into aggregates. Degradation of released adenosine diphosphate (ADP) to AMP or inhibition of phosphatidylinositol 3-kinase (PI3-K) prevented this response without affecting stationary adhesion and blocked aggregation. These findings indicate that an initial signal induced by stressed GPIbalpha-VWF bonds leads to alpha(IIb)beta(3) activation sufficient to support localized platelet adhesion. Then, additional signals from ADP receptors and possibly ligand-occupied alpha(IIb)beta(3), with the contribution of a pathway involving PI3-K, amplify platelet activation to the level required for aggregation. Our conclusions modify those proposed by others regarding the mechanisms that regulate signaling between GPIbalpha and alpha(IIb)beta(3) and lead to platelet adhesion and aggregation on immobilized VWF.

Platelet–Monocyte Aggregates

Thrombus formation within a coronary vessel is the precipitating event in unstable coronary syndromes. The angiographic severity of coronary stenoses may not predict sites of subsequent acute coronary events, inasmuch as rupture of atheromatous plaque with thrombosis in relatively mildly stenosed vessels may underlie many acute coronary syndromes. The intact endothelium normally prevents platelet activation, but the intimal injury associated with endothelial denudation and plaque rupture exposes subendothelial collagen and von Willebrand factor, which support prompt platelet adhesion and activation. Circulating platelets can adhere either directly to collagen or indirectly, via the binding of von Willebrand factor, to the glycoprotein Ib/IX complex. Local platelet activation promotes thrombus formation and additional platelet recruitment by supporting cell surface thrombin formation and releasing potent platelet agonists, such as adenosine 5′-diphosphate, serotonin, and thromboxane A2. The central role of platelet activation in acute coronary syndromes is supported by increased platelet-derived thromboxane and prostaglandin metabolites detected in patients with acute coronary syndromes1 and the clear clinical benefit of treatment with aspirin for prevention of acute coronary events.2 See p 2166 The importance of platelet-dependent thrombosis has made the platelet aggregate a common therapeutic target in acute coronary syndromes. Recently, such therapies have included the use of aspirin, thienopyridines, and direct glycoprotein IIb/IIIa inhibitors. Although the mechanism of action of these various agents differs, they all inhibit fibrinogen-dependent platelet–platelet associations (ie, platelet aggregation). However, as highlighted by the recent disappointing clinical results of trials using oral glycoprotein IIb/IIIa inhibitors,3 merely preventing the process of platelet aggregation may not always translate into clinical efficacy. Recent data suggest that patients with acute coronary syndromes have not only increased interactions between platelets (homotypic aggregates) but also increased interactions between platelets and leukocytes (heterotypic aggregates) detectable in circulating blood. These aggregates form when platelets are …

Platelet glycoprotein IIb/IIIa receptors and Glanzmann's thrombasthenia.

Platelet aggregation and fibrin formation are essential for the maintenance of normal hemostasis, a system designed to act quickly and effectively to arrest hemorrhage. This system is also triggered by pathogenic events, such as the rupture of an atherosclerotic plaque, which can lead to thrombotic vaso-occlusion, ischemia, and infarction. Platelets are a major contributor to these damaging and life-threatening thrombotic phenomena because of their adhesive properties, which result in the release of soluble mediators, platelet aggregation, and enhancement of thrombin generation.1 The platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor is a key component in the pathway to platelet aggregation; consequently, this receptor has become the target for therapeutic intervention. A paradigm of this antiplatelet treatment modality is found naturally in the inherited disorder Glanzmann’s thrombasthenia. A key feature of this disease is that patients present with mucocutaneous bleeding but only rarely demonstrate spontaneous central nervous system hemorrhage,2 a feared complication of anticoagulant and antiplatelet therapy. All of the mutations that have been identified in patients with Glanzmann’s thrombasthenia result in a functional deficiency of GPIIb/IIIa receptors,2 3 and a hallmark of this disease is the absence of agonist-induced platelet aggregation. The molecular characterization of mutations causing Glanzmann’s thrombasthenia has provided a wealth of information on structure-function relations of the GPIIb/IIIa receptor. This review will briefly summarize those mutations that affect ligand-binding domains and receptor activation and present them in the context of predicted structures. More comprehensive coverage can be found in reviews discussing the structure and function of the GPIIb/IIIa receptor complex4 5 and the clinical and molecular basis of Glanzmann’s thrombasthenia.2 3 Platelets are the first line of defense in preventing blood loss from injured blood vessels via recognition and adhesion to components of the subendothelial matrix. This event is followed by formation of a platelet …

Congenital disorders of platelet signal transduction.

After injury to the blood vessel, platelets adhere to the exposed subendothelium by a process (adhesion) that involves the interaction of a plasma protein, von Willebrand factor (vWF), and a specific protein on the platelet surface, glycoprotein Ib (GPIb; the Figure⇓). Adhesion is followed by recruitment of additional platelets that form clumps, a process called aggregation (cohesion). This involves binding of fibrinogen to specific platelet surface receptors—a complex comprising glycoproteins IIb-IIIa (GPIIb-IIIa). Activated platelets release the contents of their granules (secretion or release reaction), such as ADP and serotonin from dense granules, which subsequently cause recruitment of additional platelets. In addition, platelets play a major role in coagulation mechanisms; several key enzymatic reactions occur on the platelet membrane–lipoprotein surface. A number of physiological agonists interact with specific receptors on the platelet surface to induce responses, including a change in platelet shape from discoid to spherical, aggregation, secretion, and thromboxane A2 (TxA2) production. Other agonists such as prostacyclin inhibit these responses. Ligation of the platelet receptors initiates the production or release of several intracellular messenger molecules, including Ca2+ ions, products of phosphoinositide (PI) hydrolysis by phospholipase C (PLC; diacylglycerol [DG] and inositol 1,4,5-triphosphate [InsP3]), TxA2, and cyclic nucleotides (cAMP; the Figure⇓). These subsequently induce or modulate the various platelet responses of Ca2+ mobilization, protein phosphorylation, aggregation, secretion, and liberation of arachidonic acid. The interaction between the agonist receptors and the key intracellular effector enzymes (eg, PLA2, PLC, adenylyl cyclase) is mediated by a group of GTP-binding proteins that are modulated by GTP. As in most secretory cells, platelet activation results in …

Structural determinants within platelet glycoprotein Ibα involved in its binding to von Willebrand factor

Platelet adhesion to subendothelial structures upon injury to a vessel wall is one of the first steps in a sequence of reactions critical for the formation of a haemostatic plug, or in diseased vessels for the development of an arterial thrombus. This adhesion process is mediated by an interaction between the glycoprotein (GP) Ib - V - IX complex on the platelet surface with von Willebrand Factor (vWF), associated with collagen on the subendothelial surface. After this initial adhesion, platelets will activate, resulting in recruitment of additional platelets and adherence to each other to form the platelet plug or developing thrombus. Several studies to date have attempted to identify the regions of the GPIb - V - IX complex that are critical for binding to vWF. The vWF binding site is contained in the 45 kDa N-terminal domain of the GPIb α chain. This N - terminal domain is characterized by a structural motif consisting of 7 leucine - rich repeats (LRRs), followed by a double disulphidebonded loop and an anionic sulphated region. This review summarizes recent research efforts elucidating the characteristics of the GPIb - vWF interaction. Potential mechanisms that regulate the GPIb - vWF function are discussed, and advances in identifying functional sequences within GPIb α involved in the binding to vWF are reviewed.

Applications of a hollow fiber device to monitor hemostasis

A global assessment of hemostasis can be determined by the perfusion of unanti-coagulated whole blood through a hollow fiber at a nominal shear rate of 300 sec −1. Once the pressure across the hollow fiber is stable, uniform holes are punched through the top and bottom of the fiber, resulting in an optically detectable leakage of blood from the hollow fiber and a pressure drop throughout the system. Changes in the shear rate and flow profile at the puncture site are sufficient to activate platelets mechanically, resulting in a platelet plug observable by electron microscopy. Fibrin is the principal component of the luminal surface adjacent to the puncture site and a prominent fibrin tail extends downstream from this site. The time from the introduction of the punch site to 90% recovery of the baseline pressure defines the in vitro bleeding time (IVBT). The rate of the pressure recovery appears to be related to the efficiency of the thrombin-mediated recruitment of additional platelets to the plug. Pressure recovery can be delayed by either platelet or thrombin inhibitors.

Nitric oxide released from activated platelets inhibits platelet recruitment.

Vessel injury and thrombus formation are the cause of most ischemic coronary syndromes and, in this setting, activated platelets stimulate platelet recruitment to the growing thrombus. Recently, a constitutive nitric oxide synthase (NOS) has been identified in human platelets. To further define the capacity of platelets to produce nitric oxide (NO), as well as to study the role of this NO in platelet recruitment, we adapted a NO-selective microelectrode for use in a standard platelet aggregometer, thereby permitting simultaneous measurement of platelet aggregation and NO production. Treatment of platelets with the NO synthase inhibitor -NG-nitroarginine methyl ester (L-NAME), reduced NO production by 92+/-8% in response to 5 microM ADP compared to control but increased aggregation by only 15+/-2%. In contrast, L-NAME had a more pronounced effect on platelet recruitment as evidenced by a 35+/-5% increase in the extent of aggregation, a 33+/-3% decrease in cyclic GMP content, and a 31+/-5% increase in serotonin release from a second recruitable population of platelets added to stimulated platelets at the peak of NO production. To study platelet recruitment accurately, we developed an assay that monitors two platelet populations simultaneously. Nonbiotinylated platelets were incubated with L-NAME or vehicle and activated with ADP. At peak NO production, biotinylated platelets were added. As measured by three-color flow cytometry, there was a 56+/-11% increase in the number of P selectin- positive platelets in the nonbiotinylated population treated with L-NAME as compared to control. When biotinylated platelets were added to the L-NAME-treated nonbiotinylated population, the number of P selectin positive biotinylated plate-lets increased by 180+/-32% as compared to biotinylated platelets added to the control. In summary, stimulated platelets produce NO that modestly inhibits platelet activation but markedly inhibits additional platelet recruitment. These data suggest that platelet-derived NO may regulate platelet recruitment to a growing thrombus.

The pathophysiology of bleeding disorders presenting as abnormal uterine bleeding

Abnormal uterine bleeding is often the presenting complaint in women with underlying coagulopathies. A clear understanding of the pathophysiology of common bleeding disorders will help the practicing obstetrician/gynecologist in the diagnosis and treatment of these conditions. The normal hemostatic process can be divided into three phases. The first phase, primary hemostasis, consists of platelet adhesion and aggregation. After vascular injury, proteins in the subendothelium are exposed that promote platelet adhesion. Platelet adhesion is uniquely dependent on von Willebrand factor, a plasma protein that serves as a molecular bridge between components of the vessel wall and the platelet glycoprotein Ib/IX receptor. Activation of the adherent platelets promotes additional platelet recruitment, culminating in the formation of the platelet plug. Quantitative or qualitative defects in either the platelet or von Willebrand factor (von Willebrand disease) lead to defective primary hemostasis. Patients present with a prolonged bleeding time and mucocutaneous bleeding manifestations. In the next phase, secondary hemostasis, the plasma coagulation factors are sequentially activated, which leads to fibrin formation and cross-linking. These reactions take place primarily on the surface of activated platelets and are essential in maintaining the stability of the initial platelet plug. Defective secondary hemostasis arises from congenital or acquired deficiencies in coagulation factors. Although these defects are most often associated with bleeding into joints and soft tissues, other manifestations, including abnormal uterine bleeding, may be present. The prothrombin time and the activated partial thromboplastin time serve as initial screening tests for these coagulation disorders, although more specific tests, including factor levels, thrombin time, clot solubility, and mixing studies, are needed to fully define the defect. In the final phase of normal hemostasis, fibrinolysis, the fibrin clot undergoes an orderly process of degradation. Deficiencies in the normal inhibitors of fibrinolysis, such as α2-antiplasmin or plasminogen activator inhibitor-1, may be underdiagnosed causes of delayed bleeding because they are not identified by the usual coagulation screening tests. Disorders of primary hemostasis, including thrombocytopenia and von Willebrand disease, are particularly important to consider when evaluating women with abnormal uterine bleeding. Patients with acquired or congenital deficiencies of either coagulation factors or the regulators of the fibrinolytic system may also present with menorrhagia. Accurate diagnosis of a bleeding disorder is essential to the design of an appropriate therapeutic regimen and is likely to have important clinical implications beyond that of the presenting gynecologic complaint. (Am J Obstet Gynecol 1996;175:770-7.)

Platelet active concentration profiles near growing thrombi. A mathematical consideration

When blood contacts foreign material surfaces, platelets usually adhere and form aggregates on those surfaces, generating mural thrombi. The mechanism of mural thrombogenesis is not completely understood, but one hypothesis states that the local release of certain platelet-active substances from the platelets composing an initial small thrombus stimulates additional platelet recruitment to that thrombus, resulting in growth of the cell aggregate. The purpose of this paper is to investigate the feasibility of this hypothesis. Concentration profiles of adenosine diphosphate (ADP), thromboxane A2 (TxA2), and thrombin were computed in the vicinity of growing model thrombi 10 and 20 micron long. Wall shear rates of 100, 500, and 1,500 s-1 were considered for blood flowing through a thin rectangular slit 200 micron wide coated with collagen, a predominant subendothelial protein. The local concentrations of ADP and TxA2 were marginally large enough to stimulate platelet activation individually, while local thrombin levels can be much greater than required for stimulation. Antithrombin III, a natural thrombin inhibitor, did not significantly reduce the thrombin concentrations, but antithrombin III accelerated by heparin greatly reduced the local thrombin concentrations. The reduced thrombin levels may, however, still be large enough to activate platelets.

Platelets and their membranes in hemostasis: physiology and pathophysiology.

Platelets form a plug and promote thrombin generation at sites of vascular injury. These processes are initiated by interaction of the platelet plasma membrane with various substances within or accumulating at the injured vessel. Thus, platelet adhesion to exposed subendothelium requires the binding of von Willebrand factor to platelets. Agonists such as thrombin bind to membrane receptors, thereby stimulating the binding of fibrinogen to platelets and resulting in the aggregation of platelets onto those already adherent to the vessel wall. Agonists also stimulate transfer of membrane=bound calcium into the cytoplasm. This triggers the secretion of granule substances and results in the recruitment of additional platelets to the hemostatic plug. Concomitant with secretion, the platelet surface supports several reactions leading to thrombin generation. Thus, hemostasis requires a series of coordinated responses involving platelet membranes. A defect in any of these responses can lead to a bleeding diathesis.