Reduced platelet adhesion in flowing blood to fibrinogen by alterations in segment gamma316-322, part of the fibrin-specific region

The interaction of platelets with fibrinogen is a key event in the maintenance of a haemostatic response. It has been shown that the 12-carboxy-terminal residues of the gamma-chain of fibrinogen mediate platelet adhesion to immobilized fibrinogen. These studies, however, did not exclude the possibility that other domains of fibrinogen are involved in interactions with platelets. To obtain more insight into the involvement of other domains of fibrinogen in platelet adhesion, we studied platelet adhesion in flowing blood to patient dysfibrinogen Vlissingen/Frankfurt IV (V/FIV), to several variant recombinant fibrinogens with abnormalities in the gamma-chain segments gamma318-320 and gamma408-411. Perfusion studies at physiological shear rates showed that platelet adhesion was absent to gammaDelta408-411, slightly reduced to the heterozygous patient dysfibrinogen V/FIV and strongly reduced to the homozygous recombinant fibrinogens: gammaDelta319-320, gamma318Asp-->Ala and gamma320Asp-->Ala. Furthermore, antibodies raised against the sequences gamma308-322 and gamma316-333 inhibited platelet adhesion under shear conditions. These experiments indicated that the overlapping segment gamma316-322 contains amino acids that could be involved in platelet adhesion to immobilized fibrinogen under flow conditions. In soluble fibrinogen, this sequence is buried inside the fibrinogen molecule and becomes exposed after polymerization. In addition, we have shown that this fibrin-specific sequence also becomes exposed when fibrinogen is immobilized on a surface.     

Normal haemostasis and its regulation

Regulation of normal haemostasis and blood flow involves complex interactions between plasma proteins and blood cells, including platelets, leukocytes and the endothelial lining of blood vessels. Thrombin acts as a pivot in the maintenance of the haemostatic balance; the vascular endothelial cell in particular limits the generation of thrombin by localisation of anticoagulant processes on its luminal membrane. The endothelial cell synthesises key molecules in this process and also binds exogenously derived molecules, as well as releasing proteins of the fibrinolysis cascade. The thromboresistance of the luminal surface is further regulated by lipoxygenase and cyclo-oxygenase metabolites of unsaturated fatty acids synthesised by the endothelial cell. In response to trauma, inflammatory reactions, normal wound healing and in association with a variety of disease states, the anticoagulant and fibrinolytic mechanisms are downregulated and the procoagulant and thrombotic mechanisms predominate with resultant generation of thrombin, fibrin clot formation and subsequent platelet adhesion and aggregation. Pro-inflammatory and prothrombotic cytokines downregulate the fibrinolytic and activated protein C pathways as well as inducing synthesis of specific procoagulant and prothrombotic mediators by platelets and leukocytes as well as endothelium.     

Plasmic degradation of fibrin rapidly decreases platelet adhesion and spreading

Plasmin cleaves fibrin at or near sites involved in platelet recognition and may modulate platelet adhesion and spreading. Using an in vitro system, we have characterized the effects of limited plasmic degradation of polymerized fibrin on platelet adhesion and spreading. As shown by scanning electron microscopy, exposure to plasmin changed the tight fibrillar fibrin surface to a less dense structure with irregular and broken fibers. There was a gradient of proteolytic degradation through the fibrin clot as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis with the most extensive degradation at the surface. Plasmic degradation resulted in a rapid and progressive decrease in platelet adhesion. Plasmin exposure for 5 minutes resulted in only 6% solubilization of the fibrin but a 56% decrease in platelet adhesion. After 30 minutes of plasmin exposure, spreading of adherent platelets on fibrin also decreased sharply to a minimum of 35% of baseline. Inhibition experiments with specific monoclonal antibodies (MoAbs) indicated that platelet adhesion to undergraded fibrin involved residues within the sequence 566 through 580 of the alpha chain (including the RGDS site), the carboxyl terminal dodecapeptide of the gamma chain, and the amino terminus of the beta chain. MoAb 7E3, reactive with alpha IIb beta 3, inhibited platelet adhesion to fibrinogen by 90% +/- 5%, and to desA fibrin, prepared with Reptilase (American Diagnostica, Greenwich, CT), by 94% +/- 6%, whereas inhibition of adhesion to undegraded desAB fibrin was significantly less (48% +/- 8%, P < .01). The addition of 7E3 to MoAb T2G1, reactive with beta 15-21, significantly increased inhibition to desAB fibrin to 69% +/- 6% (P < .025), suggesting that the newly exposed amino terminus of the beta chain contributes to platelet adhesion. The results show that plasmin exposure of fibrin markedly decreases platelet adhesion and spreading, suggesting that plasmin degradation may play a role in modulating cellular responses to fibrin.     

The haemostatic plug in haemophilia A: a morphological study of haemostatic plug formation in bleeding time skin wounds of patients with severe haemophilia A

Haemostatic plug formation in four patients with severe haemophilia A (VIII:C less than 1%) was studied in skin biopsies taken at 3, 10 and 30 min and 2 h after a template bleeding time wound had been made. The primary haemostatic plug showed relatively minor changes, consisting of a delay in platelet degranulation and interdigitation. Some platelet aggregates not attached to vessels were encountered in the wound. Subsequently the primary haemostatic plug changed into a firm stable degranulated mass of interdigitated platelets. The major abnormality occurred during the fibrinous transformation. At 2 h many haemostatic plugs consisted of a thin peripheral layer of fibrin and platelet remnants around a central area containing red and white blood cells with a varying amount of plasma and only relatively few fibrin fibres. These observations suggest that fibrin formation in the periphery of the plug is less dependent of factor VIII than in central areas. The lack of fibrin formation in the centre of the plug compensating for the platelet lysis at 2 h may have caused the central erosion of the plug.     

Dynamics of leukocyte-platelet adhesion in whole blood.

The dynamics of leukocyte-platelet adhesion and platelet-platelet interaction in whole blood are not well understood. Using different platelet agonists, we have studied the whole blood kinetics of these heterotypic and homotypic interactions, the relative abilities of different leukocyte subsets to participate in platelet adhesion, and the ligands responsible for adhesion. When platelet aggregation was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide, thrombin stimulation of whole blood resulted in platelet expression of granule membrane protein 140 (GMP-140) and, simultaneously, a marked increase in the percentage of monocytes and neutrophils (PMN) binding platelets, as well as an increase in the number of platelets bound per monocyte and PMN. Lymphocytes were unaffected. Monocytes bound more platelets and at an initially faster rate than PMN. This increase in monocyte and PMN adhesion to platelets was completely inhibited by the blocking monoclonal antibody (MoAb), G1, to GMP-140. When the combination of epinephrine and adenosine diphosphate (epi/ADP) was used as a less potent agonist in the presence of RGDS, GMP-140 expression per platelet was less, and while monocyte-platelet conjugates formed, PMN-platelet conjugates did not. With epi/ADP in the absence of RGDS, there was an immediate, marked decrease in the percentage of all leukocytes with bound platelets, simultaneous with an increase in the percentage of unbound platelet aggregates. As these platelet aggregates dissociated, the percentage of monocytes and PMN with adherent platelets increased, with monocytes again binding at a faster initial rate than PMN. This recovery of monocyte and PMN adhesion to platelets was also inhibited by the G1 MoAb. We conclude that: (1) monocytes and PMN bind activated platelets in whole blood through GMP-140; (2) monocytes have a competitive advantage over PMN in binding activated platelets, particularly when less potent platelet agonists are used; and (3) platelet aggregate formation initially competes unactivated platelets off leukocytes; subsequent aggregate dissociation allows the now activated platelets to readhere to monocytes and PMN through GMP-140. These studies further elucidate the dynamic interaction of blood cells and possible links between coagulative and inflammatory processes.     

The adhesion of blood platelets on fibrinogen surface: comparison of two biochemical microplate assays

The biocompatibility of materials is frequently assessed by blood platelet adhesion, since platelet adhesion plays a considerable role in blood interaction with artificial surfaces. Blood platelets adhesion is an essential event in haemostatic and thrombotic processes. The aim of this study was to simultaneously compare simple biochemical assays widely used for evaluation of platelet static adhesion based on the determination of enzymatic activity of either lactate dehydrogenase (LDH) or acid phosphatase (ACP) in lysates of adhered platelets. Adhesion of platelets from platelet-rich plasma and washed platelets activated by either ADP or thrombin on surfaces covered with fibrinogen and well defined fibrin was studied. The results demonstrated that the amounts of adhered platelets estimated by the LDH method were significantly lower as compared with the amount obtained by ACP method. LDH but not ACP release from platelets during adhesion was shown to take place. It suggests that the LDH method should be used rather as an assay of platelet integrity. The ACP method is much more suitable for quantitative determination of platelet adhesion especially in the development and evaluation of haemocompatibility of new biomaterials.     

The effect of peptides and monoclonal antibodies that bind to platelet glycoprotein IIb-IIIa complex on the development of clot tension

The development of tension in platelet-rich clots is a manifestation of fibrin polymer binding to platelets as well as platelet contractile activity. Arg-Gly-Asp(RGD)-containing peptides of fibrinogen alpha- chain and gamma-400-411 of fibrinogen gamma chain increased clot tension considerably, especially when it developed under isometric conditions. Morphometry revealed increased confluence of oriented fibrin and platelet aggregates. Monoclonal antibodies directed against different epitopes on the glycoprotein IIb-IIIa complex had varying effects on clot tension development. Monoclonal antibodies A2A9 and 7E3 inhibited clot tension while T10 and 10E5 increased it. Since neither peptides nor antibodies affected the platelet actomyosin ATPase activity, their effect on tension must reflect the interaction between platelets and polymerizing fibrin. We conclude that gamma-400-411 and RGD-peptides increase platelet-polymerizing fibrin interaction. This suggests that clot tension requires a platelet receptor for polymerizing fibrin, which is different from the fibrinogen receptor domain required for aggregation. The results with the monoclonal antibodies support this hypothesis.     

Adhesion of platelets to surface-bound fibrinogen under flow

After platelet activation, fibrinogen mediates platelet-platelet interactions leading to platelet aggregation. In addition, fibrinogen can also function as a cell adhesion molecule, providing a substratum for adhesion of platelets and endothelial cells. In this report, we studied the adhesion of platelets to surface-immobilized fibrinogen under flow in different shear rates. Heparinized whole blood containing mepacrine-labeled platelets was perfused for two minutes at various wall shear rates from 250 to 2,000 s-1 in a parallel plate flow chamber. The number of adherent fluorescent platelets was quantitated every 15 seconds with an epifluorescent videomicroscope and digital image processing system. When compared with platelet adhesion and aggregation seen on glass surfaces coated with type I bovine collagen, a significant increase in platelet adhesion was observed on immobilized fibrinogen up to wall shear rates of 800 s-1. The adherent platelets formed a single layer on fibrinogen-coated surfaces. Under identical conditions, no significant adhesion was observed on fibronectin- or vitronectin-coated surfaces. Although platelet adhesion to collagen was substantially inhibited by the platelet inhibitors prostaglandin E1 and theophylline, these inhibitors had no effect on platelet adhesion to fibrinogen. Platelets adhered to recombinant homodimeric wild-type (gamma 400-411) fibrinogen, but not to the recombinant homodimeric gamma' variant of fibrinogen. Platelet adhesion to recombinant fibrinogen with RGD to RGE mutations at positions alpha 95-97 and alpha 572-574 was similar to that with plasma-derived fibrinogen. These results show that platelets adhere to fibrinogen-coated surfaces under moderate wall shear rates, that the interaction is mediated by the fibrinogen 400-411 sequence at the carboxy-terminus of the gamma chain, and that the interaction is independent of platelet activation and the RGD sequences in the alpha chain.     

ADP-induced platelet aggregation depends on the conformation or availability of the terminal gamma chain sequence of fibrinogen. Study of the reactivity of fibrinogen Paris 1

Fibrinogen Paris I contains a mutant gamma chain that is longer than the normal chain, resulting in altered fibrin polymerization and cross- linking. Because these functions involve the carboxy-terminal region of the gamma chain, we decided to determine whether fibrinogen Paris I or the isolated Paris I gamma chain supports normal ADP-induced platelet aggregation, a function that requires the ultimate 12 residues of the normal gamma chain (400 through 411). Aggregation of ADP-stimulated normal platelets was defective with fibrinogen Paris I and markedly depressed with the gamma Paris I chain. These findings prompted us to characterize the carboxy-terminal structure of the region of the gamma Paris I chain responsible for this activity. The carboxy-terminal cyanogen bromide (CNBr) peptide of the normal gamma chain (385 through 411) or that from gamma Paris I was isolated by differential adsorption to triethylene-tetramine resin or by reverse-phase high-performance liquid chromatography (HPLC). The CNBr peptide from the Paris I gamma chain was identical to that of the normal gamma chain in its retention time on HPLC, its amino acid composition, and its sequence. Thus, the primary structure of the gamma Paris I chain from residue 384 through 411 is normal, indicating that a peptide insertion has occurred upstream from residue 384, resulting in an impairment of those physiologic functions attributable to the carboxy-terminal end of the gamma chain from position 384 (ie, cross-linking, ADP-induced platelet aggregation, and at least a portion of the gamma chain polymerization site). These observations demonstrate that the gamma chain platelet recognition site in the fibrinogen molecule is necessary but not alone sufficient to support normal ADP-induced platelet aggregation. There appears to be an additional requirement for normal conformation of the gamma chain or availability of its terminal sequence during the interaction of fibrinogen with platelets.     

Evidence that fibrin alpha-chain RGDX sequences are not required for platelet adhesion in flowing whole blood

The role of the RGDX putative receptor-recognition sites, which are present on the alpha chains of fibrin, in promoting platelet adhesion has been examined in flowing whole blood using the rectangular perfusion chamber at wall shear rates of 340 and 1,600/s. Platelets adhered to a comparable extent to surfaces coated with native fibrin and surfaces coated with fragment X-fibrin, a product of limited fibrinolysis that lacks the RGDS sites normally present at positions 572 to 575 of the alpha chains. The strengths of these adhesive interactions were comparable based on the concentrations of the antiadhesive peptide D-RGDW required to block platelet deposition to native and fragment X-fibrin at both low and high wall shear rate. Blocking either or both RGDX sequences with peptide-specific monoclonal antibodies did not inhibit platelet deposition in perfusion experiments performed with normal blood at 340/s, indicating that neither RGD motif is required for adhesion. However, adhesion was partly inhibited by anti-RGDX antibodies when perfusions were performed with blood from an afibrinogenemic patient, suggesting the RGDX sequences may play a limited role in platelet deposition. Exposure of fibrin surfaces to plasminogen/tissue-type plasminogen activator did cause a time- dependent loss of adhesiveness, but this effect was only weakly correlated with proteolysis of the fibrin alpha chains. These observations provide evidence that neither RGDX sequence is required for platelets to adhere avidly to fibrin in flowing blood. These results further suggest that incomplete fibrinolysis yields a highly thrombogenic surface.     

Role of dystrophins and utrophins in platelet adhesion process

Platelets are crucial at the site of vascular injury, adhering to the sub-endothelial matrix through receptors on their surface, leading to cell activation and aggregation to form a haemostatic plug. Platelets display focal adhesions as well as stress fibres to contract and facilitate expulsion of growth and pro-coagulant factors contained in the granules and to constrict the clot. The interaction of F-actin with different actin-binding proteins determines the properties and composition of the focal adhesions. Recently, we demonstrated the presence of dystrophin-associated protein complex corresponding to short dystrophin isoforms (Dp71d and Dp71) and the uthophin gene family (Up400 and Up71), which promote shape change, adhesion, aggregation, and granule centralisation. To elucidate participation of both complexes during the platelet adhesion process, their potential association with integrin beta-1 fraction and the focal adhesion system (alpha-actinin, vinculin and talin) was evaluated by immunofluorescence and immunoprecipitation assays. It was shown that the short dystrophin-associated protein complex participated in stress fibre assembly and in centralisation of cytoplasmic granules, while the utrophin-associated protein complex assembled and regulated focal adhesions. The simultaneous presence of dystrophin and utrophin complexes indicates complementary structural and signalling mechanisms to the actin network, improving the platelet haemostatic role.     

Vascular endothelial growth factor-stimulated endothelial cells promote adhesion and activation of platelets

Coagulation abnormalities, including an increased platelet turnover, are frequently found in patients with cancer. Because platelets secrete angiogenic factors on activation, this study tested the hypothesis that platelets contribute to angiogenesis. Stimulation with vascular endothelial growth factor (VEGF, 25 ng/mL) of human umbilical vein endothelial cells (HUVECs) promoted adhesion of nonactivated platelets 2.5-fold. In contrast, stimulation of HUVECs with basic fibroblast growth factor (bFGF) did not promote platelet adhesion. By blocking tissue factor (TF) activity, platelet adhesion was prevented and antibodies against fibrin(ogen) and the platelet-specific integrin, alpha(IIb)beta(3), inhibited platelet adhesion for 70% to 90%. These results indicate that VEGF-induced platelet adhesion to endothelial cells is dependent on activation of TF. The involvement of fibrin(ogen) and the alpha(IIb)beta(3) integrin, which exposes a high-affinity binding site for fibrin(ogen) on platelet activation, indicates that these adhering platelets are activated. This was supported by the finding that the activity of thrombin, a product of TF-activated coagulation and a potent platelet activator, was required for platelet adhesion. Finally, platelets at physiologic concentrations stimulated proliferation of HUVECs, indicative of proangiogenic activity in vivo. These results support the hypothesis that platelets contribute to tumor-induced angiogenesis. In addition, they may explain the clinical observation of an increased platelet turnover in cancer patients. Platelets may also play an important role in other angiogenesis-dependent diseases in which VEGF is involved, such as diabetes and autoimmune diseases. (Blood. 2000;96:4216-4221)     

Minimal platelet deposition and activation in models of injured vessel wall ensure optimal neutrophil adhesion under flow conditions.

Platelets at injured vessel wall form an adhesive surface for leukocyte adhesion. The precise relation between platelet adhesion and activation and leukocyte adhesion, however, is not known. We therefore used various models of injured vessel wall to form different patterns of platelet adhesion. The interaction of polymorphonuclear neutrophils (PMNs) was subsequently studied under flow conditions. In the absence of platelets, not only endothelial cell, smooth muscle cell, and fibroblast matrices but also purified matrix proteins (fibrinogen, collagen, and fibronectin) barely support PMN adhesion. The presence of platelets, however, strongly enhances PMN adhesion. PMN adhesion shows a proportional increase with platelet coverage up to 15%. Although PMNs roll over the scarcely scattered platelets, they speed up again when encountering surfaces without platelets. This "hopping" interaction of PMNs vanishes with platelet coverage >15%. Unobstructed rolling of PMNs is than observed and soon leads to a maximal adhesion of 1000 to 1200 cells/mm2. The mean rolling velocity of PMNs continues to decrease with higher platelet coverage. Platelet aggregate formation is an accepted indicator of platelet activation. The presence of platelet aggregates instead of contact or spread platelets, however, does not increase PMN adhesion. Also, additional stimulation of surface-associated platelets by thrombin fails to influence PMN adhesion. Moreover, indomethacin as an inhibitor of platelet activation and aggregation does not change the subsequent PMN interaction. In conclusion, approximately 15% of platelet coverage is sufficient for optimal PMN adhesion. Increasing platelet coverage increases the availability of platelet-associated receptors that lower PMN rolling velocity. Additional activation of adherent platelets makes no difference in the expression of relevant adhesion receptors. Therefore, minimal vascular damage in vivo and only scarce platelet adhesion will already evoke significant colocalization of leukocytes.     

On the role of von Willebrand factor in promoting platelet adhesion to fibrin in flowing blood

Platelet adhesion to fibrin at high shear rates depends on both the glycoprotein (GP) IIb:IIIa complex and a secondary interaction between GPIb and von Willebrand factor (vWF). This alternative link between platelets and vWF in promoting platelet adhesion to fibrin has been examined in flowing whole blood with a rectangular perfusion chamber. Optimal adhesion required both platelets and vWF, as shown by the following observations. No binding of vWF could be detected when plasma was perfused over a fibrin surface or when coated fibrinogen was incubated with control plasma in an enzyme-linked immunosorbent assay. However, when platelets were present during perfusion, interactions between vWF and fibrin could be visualized with immunoelectron microscopy. Exposure of fibrin surfaces to normal plasma before perfusion with severe von Willebrand's disease blood did not compensate for the presence of plasma vWF necessary for adhesion. vWF mutants in which the GPIIb:IIIa binding site was mutated or the GPIb binding site was deleted showed that vWF only interacts with GPIb on platelets in supporting adhesion to fibrin and not with GPIIb:IIIa. Complementary results were obtained with specific monoclonal antibodies against vWF. Thus, vWF must first bind to platelets before it can interact with fibrin and promote platelet adhesion. Furthermore, only GPIb, but not GPIIb:IIIa is directly involved in this interaction of vWF with platelets.     

Sticky and promiscuous plasma proteins maintain the equilibrium between bleeding and thrombosis

A vascular fissure requires a patch that must be provided by constituents of the cellular and fluid phases of flowing blood. The principal components involved in primary haemostasis are platelets, collagen and von Willebrand factor (vWF). Platelets, the cellular elements of the patch, are inert until they encounter conditions that trigger their activation. Platelet adhesion and aggregation at the site of vascular injury lead to the formation of a platelet plug and to a local activation of the coagulation cascade. The resulting final product of blood coagulation is a fibrin network that stabilises the primary platelet plug. Most coagulation factors are zymogens of serine proteases. They are converted from an inactive form to an active enzyme by limited proteolytic cleavage of one or a few peptide bonds. The coagulation reactions must become extinguished as soon as the patch in the injured blood vessel has been established. Several inhibitors, present in excess in plasma, neutralise the surplus of remaining proteases, and the fibrinolytic system dissolves the plug after the surrounding tissue has been repaired. In fulfilling their function to control the fluidity and integrity of the vascular system, the plasmatic and cellular haemostatic players undergo multiple interactions of two kinds: they recognize and bind, often irreversibly, to several partners which are present in their immediate environment. On the other hand, some haemostatic factors, such as fibrinogen and von Willebrand factor, enhance their stickiness by polymerisation of identical subunits carrying multiple adhesive sites. Several haemostatic plasma proteins and their cellular surface receptors are involved in or may be affected by other homeostatic systems, such as immune response, complement activation, cytokine release, cell proliferation, growth and differentiation. These diverse functions are only possible because of the modular structure of participating proteins. In the process of evolution a series of structural modules have been incorporated into protein molecules as their integral domains by exon duplication and shuffling. Owing to variable conformations of the resulting multi-domain proteins, the same modules may perform different tasks and be recognized only by specific ligands, thus controlling the delicately balanced system of haemostasis.     

Activated and unactivated platelet adhesion to monocytes and neutrophils.

To examine the possible receptor-ligand pairs mediating adhesion of activated and "unactivated" platelets to leukocytes and the kinetics of leukocyte-platelet binding, we developed a flow cytometric assay using isolated cell fractions to accurately measure heterotypic cell adhesion, including both total leukocyte-platelet conjugate formation as well as the number of platelets bound per leukocyte. We have shown that (1) activated platelet binding to both polymorphonuclear leukocytes (PMN) and monocytes is dependent on both a specific epitope (blocked by monoclonal antibody G1) of granule membrane protein-140 (GMP-140) and the presence of divalent cations; (2) unactivated platelets bind to 87% of viable, resting monocytes but to only 34% of PMN; (3) the receptor(s) on unactivated platelets that mediate adhesion to PMN and monocytes do not require divalent cations and become nonfunctional after thrombin activation; and (4) the kinetics of platelet adhesion to monocytes and PMN indicate that monocyte adhesion is favored over neutrophil adhesion. We conclude that platelet-heterotypic cell adhesion is a dynamic process reflecting the activation status of the platelet and differential binding abilities of leukocytes.     

Characterization of the gamma chain platelet binding site on fibrinogen fragment D.

Glycoprotein (GP) IIb/IIIa on adenosine diphosphate (ADP)-activated human platelets interacts with specific sites on the fibrinogen molecule leading to aggregation. We characterized the platelet-binding site on the gamma chains of fibrinogen using plasmic fragments D gamma A and D gamma'. Fragment D gamma A, which contains the carboxy terminal gamma A400-411 platelet-binding sequence (HHLGGAKQAGDV), was 70-fold more active than the synthetic gamma A400-411 peptide in inhibiting ADP-induced platelet aggregation. Fragment D gamma A inhibited fibrinogen binding and also bound directly to ADP-activated platelets. The Kd values determined for fibrinogen and fragment D gamma A binding were 0.55 mumol/L and 1.2 mumol/L, respectively. In contrast, fragment D gamma', which differs from fragment D gamma A with respect to its gamma chain sequence from position 408 to the COOH-terminus at position 427, did not inhibit platelet aggregation or fibrinogen binding, and did not bind directly to the platelet surface. Denaturation of fragment D gamma A with guanidine-HCl caused a loss of inhibitory activity in platelet aggregation assays. These data indicate that the native conformation of the gamma chain platelet-binding site on fibrinogen is important for optimal binding to GPIIb/IIIa.     

The molecular mechanism of platelet adhesion

One of the most primitive of host-defence mechanisms is haemostasis, the ability to control blood loss. In response to vascular trauma, platelets rapidly adhere to the exposed subendothelial matrix, a process that ultimately results in the sealing of the vessel by a plug of platelets stabilised by fibrin. Paradoxically, it is the same cascade of events that leads to thrombosis and vessel occlusion, resulting in heart attack and stroke. The molecular events involved in platelet adhesion have therefore been the subject of intense investigation. In all but the largest blood vessels, the initial contact adhesion of platelets is mediated by subendothelial matrix bound von Willebrand Factor (v WF) and a specific v WF receptor on platelets, the glycoprotein (GP) Ib- V-IX complex. Our understanding of this process arose from analysis of two congenital bleeding disorders, von Willebrand's disease and the Bernard-Soulier syndrome, in which v WF or the GP Ib- V-IX, respectively, are either absent or dysfunctional. This overview discusses our current molecular understanding of platelet adhesion and how engagement of v WF by the GP lb- V-IX complex on platelets initiates the subsequent events in platelet activation leading to either haemostasis or thrombosis.     

Platelet/polymorphonuclear leukocyte interaction in dynamic conditions: evidence of adhesion cascade and cross talk between P-selectin and the beta 2 integrin CD11b/CD18

Adhesion between platelets and polymorphonuclear leukocytes (PMN) is a key event in thrombosis and inflammation. Double color fluorescence- activated cell sorter (FACS) analysis was used to determine the extent and kinetics of adhesion of thrombin-activated platelets to resting or activated PMN when mixed cell populations were incubated in dynamic conditions. Activated platelets bound very rapidly to PMN. Mixed cell conjugates reached a maximum at 1 minute and were reversible within 10 minutes. Platelet/PMN adhesion required both Ca2+ and Mg2+ and was markedly increased by the presence of Mn2+. The latter made mixed cell conjugates stable up to 10 minutes. Adhesion of platelets required metabolic activity of PMN and was abolished by tyrosine kinase inhibitors. Furthermore, adhesion of platelets to PMN resulted in binding of a monoclonal antibody (MoAb 24) known as beta 2 integrins "activation reporter." When PMN were activated by exogenous stimuli, the adhesion of platelets was markedly increased: fMLP induced a rapid and transient effect, while PMA resulted in a slower, but stable, increase in mixed conjugates formation. The hypothesis that activated PMN beta 2 integrins are able to bind a counter-receptor on platelets was directly demonstrated by the increase of mixed cell conjugates following PMN treatment with KIM127 and KIM185, two anti-CD18 antibodies able to induce the active conformation of beta 2 integrins. Consistently, two other anti-CD18, as well as an anti-CD11b inhibitory antibody abolished platelet/PMN adhesion. PMN beta 2 integrin activation was not the only mechanism for activated platelet/PMN adhesion to occur: indeed, this phenomenon could also be inhibited by two anti-P-selectin antibodies. Resting platelets did not adhere to resting PMN, but markedly adhered to fMLP- or PMA-activated PMN. Resting platelet/fMLP-activated PMN adhesion was abolished by anti-CD18 antibodies, but not by anti-P-selectin antibodies. In conclusion, activated platelet/PMN interaction can be modeled as an adhesion cascade involving a P-selectin-dependent recognition step and a functional signal. The latter proceeds through tyrosine kinase activation and enables a beta 2 integrin-dependent adhesion to a not yet identified counter-receptor constitutively expressed on platelet surface.     

Src family kinases mediate neutrophil adhesion to adherent platelets

Polymorphonuclear leukocyte (PMN)-platelet interactions at sites of vascular damage contribute to local and systemic inflammation. We sought to determine the role of "outside-in" signaling by Src-family tyrosine kinases (SFKs) in the regulation of alphaMbeta2-integrin-dependent PMN recruitment by activated platelets under (patho)physiologic conditions. Activation-dependent epitopes in beta2 integrin were exposed at the contact sites between PMNs and platelets and were abolished by SFK inhibitors. PMNs from alphaMbeta2(-/-), hck(-/-)fgr(-/-), and hck(-/-)fgr(-/-)lyn(-/-) mice had an impaired capacity to adhere with activated platelets in suspension. Phosphorylation of Pyk2 accompanied PMN adhesion to platelets and was blocked by inhibition as well as by genetic deletion of alphaMbeta2 integrin and SFKs. A Pyk2 inhibitor reduced platelet-PMN adhesion, indicating that Pyk2 may be a downstream effector of SFKs. Analysis of PMN-platelet interactions under flow revealed that SFK signaling was required for alphaMbeta2-mediated shear-resistant adhesion of PMNs to adherent platelets, but was dispensable for P-selectin-PSGL-1-mediated recruitment and rolling. Finally, SFK activity was required to support PMN accumulation along adherent platelets at the site of vascular injury, in vivo. These results definitely establish a role for SFKs in PMN recruitment by activated platelets and suggest novel targets to disrupt the pathophysiologic consequences of platelet-leukocyte interactions in vascular disease.