A mathematical model of the kinetics of platelets and plasma hemostasis system interaction.

A mathematical model of the kinetics of platelet-activated blood coagulation is presented. Non-linear positive feedbacks due to the action of co-factor Va and VIIIa, thrombin-induced platelet activation, the secretion of factor V by platelets are taken into account. The intrinsic pathway is shown not to be activated in the absence of platelets for small stimulation intensities. The activation occurs if initial platelet activation by inductors other than thrombin exceeds a threshold value.

The role of platelet prostanoids and dense granule compounds in initial attachment, spreading and aggregation of platelets on collagen substrates.

The role of platelet prostanoids, ADP and 5HT in initial attachment, spreading and aggregation of platelets on collagen substrates (CI, CIII, CIV, CV, CC) was studied. A positive linear correlation was found between thrombi-like aggregate formation on collagen substrates and production of platelet prostanoids. No correlation was established between platelet aggregation and 14C-5HT release. Thrombi-like aggregate formation was completely inhibited by indomethacin and TXA2/PGH2 antagonists (13-APA and BM 13.177). Both 13-APA and BM 13.177 had no effect on platelet spreading, while indomethacin inhibited this process by 25%. The ADP-scavenger system (CP/CPK) inhibited platelet aggregation and spreading by 25-30%. Initial attachment was not influenced by aspirin, indomethacin and CP/CPK. The data obtained indicate that platelet aggregation on collagen substrates is mediated by PGH2 and TXA2 production. These compounds slightly affect the platelet spreading. Both platelet spreading and aggregation on collagen substrates are only partially mediated by ADP and 5HT release. Initial attachment of platelets does not depend on the release reaction and PGH2/TXA2 synthesis.

Phorbol ester stimulates platelet spreading and thrombi-like aggregate formation on the surface of immobilized type V collagen.

We have studied the effect of the tumor-promoting phorbol ester, 4 beta-phorbol-12 beta-myristate-13 a-acetate (PMA), and of the stable prostaglandin endoperoxide analogue U46619 on the interaction of human blood platelets with surfaces coated with monomeric human type V collagen (CV) and on free calcium concentration in platelet cytoplasm. It was shown by scanning electron microscopy that native resting platelets sparingly attach to CV and fail to spread or aggregate on the collagenous substrate in the absence of PMA and U46619. Addition of 0.15-1.5 nM PMA or 1.5 microM U46619 stimulates platelet spreading and formation of multilayer (thrombi-like) platelet aggregates on the per se non-thrombogenic type V collagen substrate. It was further demonstrated using the fluorescent indicator quin2 that U46619 (0.1 microM) increases cytoplasmic free calcium concentration from basal level (100-120 nM) up to 600 nM, whereas PMA (0.75-15 nM) exerts only a minor effect, increasing free calcium level by 30-40 nM. These results indicate that the tumor-promoting phorbol ester PMA induces massive platelet spreading and aggregation on surfaces coated with non-thrombogenic type V collagen via activation of protein kinase C with little or no apparent change in free cytoplasmic calcium.

Effects of prostacyclin analogue (carbacyclin) on the interaction of platelets with different collagen substrates. Inhibition of cAMP increase by collagens type I, III, and IV.

We investigated the effects of a stable prostacyclin analogue, carbacyclin, on the interaction of platelets with collagen substrates differing in their ability to activate platelets: human collagens type I, III, IV and V (CI, CIII, CIV and CV), and commercial calf skin collagen type I (CSC). The total adhesion was measured using 51Cr-labelled platelets, and quantitative morphometry of adherent platelets was performed by scanning electron microscopy (SEM). Carbacyclin in the concentrations inducing a 10-fold rise in platelet cAMP did not affect the adhesion of platelets to weak substrates, CV and CSC, but reduced the adhesion to strong substrates, CIV (by 49%) and CI/CIII (by 78%), which stimulated massive spreading and formation of surface-bound aggregates respectively. Carbacyclin inhibited all morphological manifestations of platelet activation associated with adhesion: conversion of native discoid platelets to spherical ones on CSC; massive spreading on CIV; and aggregate formation on CI/CIII. Massive spreading and aggregation on a weak substrate (CSC) stimulated by arachidonic acid and thrombin was also inhibited by carbacyclin. Under the same concentration of agonists aggregation of platelets was more sensitive to the action of carbacyclin, than spreading. Strong collagen substrates CI, CIII and CIV, but not CV and gelatin, inhibited the carbacyclin-induced rise in platelet cAMP.

Effect of metabolic inhibitors on platelet attachment, spreading and aggregation on collagen-coated surfaces.

The interaction of human gel-filtered platelets (GFP) with surfaces coated with fibrillar calf skin collagen (CSC) or monomeric human type I, III, IV, and V collagen (CI, CIII, CIV, CV) includes both energy dependent and independent stages. Incubation of platelets with a collagen-coated surface at 4 degrees C versus 37 degrees C reduces only shape change and the spreading response of adhering platelets, but does not affect the initial attachment. Additionally, the energy dependence was evident from the reduction of platelet spreading and platelet aggregate formation in the presence of 2-Deoxy-D-glucose (2DG). Antimycin A (AMA), Oligomycin (OM), or 2,4-Dinitrophenol (DNP) did not abolish the adhesion-induced platelet activation, indicating that the energy is supplied by glycolysis rather than by oxydative phosphorylation. In contrast, neither inhibition of glycolysis, nor inhibition of the respiratory chain did affect the initial attachment of nonactivated platelets to the collagen-coated surface. The present data suggest (i) that during the interaction of platelets with collagenous substrates there exists an initial energy independent attachment stage, and (ii) that the following stages of adhesion-induced platelet activation require metabolic energy supported mainly by anaerobic glycolysis.

Binding of human monomeric type I collagen to platelets.

Interaction of platelets with subendothelial collagen is important in primary hemostasis and thrombosis. Although activation of platelets by collagen polymers has been widely investigated, only insufficient data are available concerning the binding of genetically distinct collagen types in their triple helical (monomeric) form to platelets. We report on the binding of 125I-labeled human type I collagen to platelets. The binding assay was performed at 20 degrees C in the presence of arginine in order to prevent polymerization of the collagen monomers. The binding of monomeric 125I-labeled human type I collagen is dose- and time-dependent, saturable and specific, since it is competitively inhibited by unlabeled type I collagen, but not by unlabeled human type V collagen. Scatchard analysis reveals a class of specific high affinity binding sites with a Kd of 2.5 X 10(-8) M. These results suggest that platelets interact with type I collagen through specific binding sites, and that there are various different binding sites on the platelet membrane for the genetically distinct collagen types.

Influence of thromboxane receptor-antagonists on the interaction of platelets with solid-phase immobilized human collagen.

The interaction of blood platelets with surfaces coated with human type I, III, IV, and V collagen (CI, CIII, CIV, and CV) has been studied. Using scanning electron microscopy it was demonstrated that the reactivity of the collagen substrates for platelets varies widely. In contrast to CV and CIV, on surfaces coated with CI and CIII, along with spreading, the formation of thrombi-like platelet aggregates occurs. Previously, it was demonstrated that this thrombogenesis in vitro correlates well with the synthesis of platelet prostanoids, and is sensitive to inhibitors of TXA2-synthesis. Therefore, we have investigated the influence of the TXA2/PGH2 receptor-antagonists, 13-azaprostanoic acid (13-APA) and the BM 13.177 compound, on the formation of thrombi-like platelet aggregates on CI and CIII-coated surfaces. It was demonstrated that both 13-APA and BM 13.177 cause a dose-dependent inhibition of the thrombogenesis without any effect on the initiation of the thrombogenesis without any effect on the initial attachment and the spreading of platelets on collagen-coated surfaces. The obtained data suggest that (1) the formation of platelet aggregates on CI and CIII-coated surfaces is triggered by platelet prostanoids; (2) TXA2/PGH2 receptor-antagonists may be useful as antithrombotic drugs.

Interaction of stable prostaglandin endoperoxide analogs U46619 and U44069 with human platelet membranes: coupling of receptors with high-affinity GTPase and adenylate cyclase.

It has been demonstrated using a membrane preparation of human platelets that stable analogs of PGH2, U46619 and U44069, control the activity of adenylate cyclase and a high-affinity hormone-sensitive GTPase. At 10(-8)-10(-6) M, the analogs inhibit the basal activity of adenylate cyclase by 20-25%. With a further rise in U46619 and U44069 concentrations up to 10(-5)-10(-4) M, the inhibition is abolished and adenylate cyclase activity is stimulated in a dose-dependent fashion. In the presence of PGE1, only inhibitory action of U46619 was observed at all the concentrations tested. The inhibitory action of the analogs on adenylate cyclase correlates with the activation of the high-affinity hormone-sensitive GTPase. It is concluded that U46619 and U44069 inhibit human platelet adenylate cyclase via specific receptors coupled to the GTP-binding inhibitory protein.

Decrease of platelet aggregation and spreading via inhibition of the cAMP phosphodiesterase by trapidil.

Trapidil (N,N-diethyl-5-methyl[1,2,4]triazolo[1,5-alpha]pyrimidine-7-amine ) inhibits platelet spreading and aggregation induced by arachidonic acid (AA), a stable analogue of prostaglandin (PG) endoperoxides (U46619), ADP, and low concentrations of thrombin, but not by A23187 and high concentrations of thrombin. Trapidil does not affect platelet adenylate cyclase but inhibits the cAMP PDE by approx. 50%. PDE inhibition proceeds via a competitive mechanism (Ki = 0.52 mM) and is not mediated by calmodulin inhibition. Trapidil does not change the platelet basal cAMP level but potentiates an increase of cAMP induced by the stable prostacyclin analogue (6 beta-PGI1). These results suggest that trapidil antiplatelet effects may be due to the inhibition of platelet PDE.

Ca2+ binding and charge movements in membranes of platelets and sarcoplasmic reticulum.

The properties of the Ca2+-pump system of platelet microsomes isolated without Ca2+-precipitating anions are studied. Passive Ca2+ binding to the microsomes takes place in a noncooperative manner with Kd = 0.7 microM. Half-maximal stimulation of ATP-dependent transport occurs at 0.4 microM Ca2+. The velocity of Ca2+ uptake, Ca2+ capacity and the level of phosphoprotein in platelet microsomes are significantly lower than in cardiac microsomes. Energization of platelet and muscle microsomes and activation of intact platelets result in opposite charge redistribution in hydrophobic regions of the membranes. It is concluded that these charge movements are caused by Ca2+ binding to and dissociation from nonpolar binding sites in the membranes.

Step-by-step analysis of adhesion of human platelets to a collagen-coated surface defect in initial attachment and spreading of platelets in von Willebrand's disease.

Adhesion of platelets from the platelet-rich plasma (PRP) of patients with von Willebrand's disease (vWD) and healthy donors has been studied in a simple model system - wells of multiwell tissue culture plates coated with fibrillar calf skin collagen (CSC). This model is characterized by: (i) the presence of only one constituent of the vessel wall connective tissue matrix (collagen), (ii) the absence of surface-bound aggregates and thrombi, (iii) absence of overlapping of neighbouring spread platelets. A morphometric quantitation of adhesion by scanning electron microscopy (SEM) has been carried out. It allows to subdivide this process into three stages: 1) initial attachment of unspread platelets to the substrate, 2) platelet spreading on the substrate, and 3) attachment of unspread platelets to the upper surface of spread platelets. It was established that the PRP of vWD patients, compared to that of healthy donors, is characterized by a decreased total adhesion of platelets to a CSC-coated surface, which is manifested in the impairment of both the initial attachment and subsequent spreading of platelets. Addition of platelet-free plasma from healthy donors to the vWD PRP completely restores platelet spreading on collagen but little affects the initial attachment. These experiments performed on isolated collagen preparations provide further evidence for the initial attachment and spreading of platelets on collagenous constituents of the subendothelium being factor VIII/von Willebrand factor (FVIII/vWF)-dependent. In contrast to the adhesion on the collagen substrate, the adhesion of platelets from vWD PRP to a foreign surface, polystyrene plastic of uncoated wells, is the same as that of the normal PRP and, thus, FVIII/vWF-independent.

Platelet prostanoids in interaction of platelets with collagen substrates. I. Activation of platelets by surfaces coated with different types of human collagen.

We have studied the interaction of human platelets with surfaces coated with human type I, III, IV, and V collagen (CI, CIII, CIV, and CV). It was established using scanning electron microscopy that the reactivity of the collagen substrates for platelets varies widely. On CV, only the initial attachment of platelets takes place; spreading actively goes on CIV while on CI and CIII, along with spreading, the formation of multilayer thrombi-like platelet aggregates occurs. The production of malondialdehyde induced by the interaction of platelets with CI and CIII substantially exceeds that stimulated by CIV and CV. Indomethacin practically completely inhibits the formation of thrombi-like aggregates but only by 25% inhibits platelet spreading. An ADP-scavenger creatine phosphate/creatine phosphokinase inhibits the formation of thrombi-like aggregates and platelet spreading by 25-30%. The obtained data demonstrate that: (i) the formation of thrombi-like aggregates on CI and CIII is mediated mainly by the synthesis of platelet prostanoids, and not by the ADP release; (ii) the spreading of platelets on CIV, CIII, and CI is only partially mediated by prostanoid synthesis and ADP release which suggests a participation of other mechanisms in this process.

Platelet prostanoids in interaction of platelets with collagen substrates. II. Effects of exogenous arachidonic acid, stable PG endoperoxides analogue, and aspirin.

The effects of (i) the exogenous arachidonic acid (AA), (ii) stable PG endoperoxide analogue U46619, and (iii) cyclooxygenase inhibitor aspirin on the interaction of platelets with a surface coated with commercial fibrillar calf skin collagen (CSC) were studied using scanning electron microscopy. AA and U46619 stimulate massive spreading of platelets on the CSC substrate and formation of the surface-bound multilayer (thrombi-like) aggregates. The stimulation of spreading and formation of thrombi-like aggregates induced by AA correlate with the TXA2 synthesis in platelets. Unlike AA, U46619 induces these processes without transformation into TXA2 and stimulation of its synthesis in platelets. Aspirin prevents the AA-induced platelet spreading, formation of the surface-bound thrombi-like aggregates, and TXA2 synthesis. In the absence of soluble platelet inducers, aspirin decreases the CSC substrate-induced disc - sphere transformation and spreading of platelets, but doesn't affect the initial attachment of platelets to the CSC substrate.

Antianginal drug trapidil (Rocornal) inhibits spreading of platelets and formation of surface-bound thrombi-like aggregates.

Effects of an antianginal drug trapidil (Rocornal) on platelet interaction with a surface coated with fibrillar calf skin collagen (CSC) have been studied by scanning electron microscopy. Gel filtered human platelets were incubated with the CSC substrate in the absence or in the presence of soluble inducers of platelet activity: arachidonic acid (AA), stable analogue of prostaglandin (PG) endoperoxides, U46619, and thrombin. In the absence of soluble inducers, trapidil does not alter the total number of adherent platelets. At the same time, trapidil inhibits the shape change of adherent platelets, induced by the CSC substrate, increasing the percentage of discoid and decreasing the percentage of spread platelets. It was demonstrated earlier (1) and in the present study that soluble inducers of platelet activity stimulate massive spreading of platelets and formation of surface-bound thrombi-like aggregates on the CSC substrate. Trapidil completely prevents the effects of the exogenous AA and U46619 on platelet-substrate interactions, but inhibits the AA-stimulated synthesis of thromboxane A2 (TXA2) in platelets by 40-50% only. Trapidil also blocks platelet aggregation in suspension, spreading and formation of surface-bound aggregates induced by low, but not high, concentrations of thrombin. Possible sites of trapidil action are discussed.

Arachidonic acid and stable analogue of prostaglandin endoperoxides (U46619) induce platelet spreading and thrombi-like aggregate formation on a collagen substrate. Effect of fluid dynamics.

Soluble plasma inducers and inhibitors of platelet activity and fluid dynamics of the blood stream are effective modulators of platelet-vessel wall interactions. Effects of platelet activity inducers, arachidonic acid (AA) and stable prostaglandin endoperoxides analogue (U46619), on platelet disposition on the bottom of multiwell tissue culture plates coated with fibrillar calf skin collagen (CSC) have been studied by scanning electron microscopy (SEM). Both agents stimulate platelet spreading and formation of large surface-bound multilayer (thrombi-like) aggregates on a CSC substrate. AA and U46619 effects on spreading and thrombi-like aggregate formation depend on the speed of platelet suspension shaking during platelet deposition on the surface. In the absence of shaking, both inducers mainly stimulate the spreading of platelets: spread platelets fuse and form widespread sheets covering up to 50% of the CSC-coated surface. An increase in the shaking speed leads to the decrease of the platelet spreading, while the number of surface-bound thrombi-like aggregates grows, reaching the maximum at a shaking speed of 40 back and forth cycles per min. The thrombi-like aggregates mainly consist of fused platelets and always contain the basal sheet of spread platelets, which suggests the participation of the latter in aggregate attachment to the surface. Large aggregates are absent in the population of nonadherent platelets. The obtained data indicate that AA metabolites participate in platelet spreading and thrombi-like aggregate formation, the processes specific for platelet-surface interactions. The use of the suggested model for the in vitro study of platelet spreading and mural thrombi formation, and for screening of antithrombotic and thrombolytic drugs is discussed.

Endothelial cell culture on fibrillar collagen: model to study platelet adhesion and liposome targeting to intercellular collagen matrix.

Human umbilical endothelial cells (ECs) were grown on fibrillar type I collagen in 16.4-mm multiwell tissue culture plates. Human platelets were added to the wells, and platelet adhesion to collagen was examined by scanning electron microscopy and radioisotopic technique in the absence of ECs and in preconfluent and confluent EC cultures. Single adherent platelets of different shapes as well as small aggregates were seen on collagen surface. Human plasma fibronectin added to the system stimulated platelet adhesion and their spreading on collagen. ECs had no effect on the percentage of platelets adherent to collagen-coated gaps in preconfluent culture but decreased the number of spread platelets. It is demonstrated that collagen-coated gaps can bind 14C-labeled liposome--antibody and 14-C-labeled liposome--fibronectin conjugates. ECs grown on fibrillar collagen are suggested as useful models for screening of antiplatelet drugs and for the study of drug targeting to the areas of vascular injury for prevention of thrombosis.

Human plasma fibronectin promotes the adhesion and spreading of platelets on surfaces coated with fibrillar collagen.

This letter reports that human plasma fibronectin stimulates the interaction of platelets with fibrillar collagen (cell adhesion) and also dramatically changes the shape of adherent platelets, promoting their spreading. The stimulating effect of fibronectin is due to its interaction with collagen fibrils since free fibronectin does not influence the adhesion or spreading of platelets. The results obtained suggest a new physiological function for plasma fibronectin in the control of platelets interaction with damaged vessel walls.