The platelet open canalicular system: a final common pathway.

Channels of the surface-connected, open canalicular system (OCS) of human platelets serve as the pathway for transport of substances into the cells and as conduits for the discharge of alpha granule products secreted during the platelet release reaction. The purpose of the present study was to determine if both functions of the OCS can take place simultaneously. Suspensions of washed platelets were exposed to thrombin at 1 U/ml for 5, 60, or 180 seconds in the presence of fibrinogen molecules coupled to particles of colloidal gold (Fgn/Au). The samples were fixed in a low concentration of glutaraldehyde and embedded in L.R. White resin to preserve antigenicity. Thin sections were exposed to a rabbit polyclonal antibody to human fibrinogen followed by an anti-rabbit IgG coupled to 5-nm gold beads. Thrombin caused Fgn/Au particles to bind to platelets and enter channels of the surface-connected OCS. Endogenous fibrinogen detected by immunogold 5-nm beads were localized to alpha granules in resting platelets and 5 seconds after thrombin stimulation. At 60 seconds and 3 minutes Fgn/Au particles were present in swollen alpha granules, as well as OCS channels. Fibrinogen gold beads were evident in alpha granules and OCS channels connected to the platelet surface. The 18- to 20-nm Fgn/Au particles were in the same channels of the OCS as fibrinogen gold beads. The OCS is a final common pathway for uptake of particulates and discharge of secretory products in thrombin-activated human platelets.     

Mobility of GPIIb-IIIa receptors within membranes of surface- and suspension-activated platelets does not depend on assembly and contraction of cytoplasmic actin

Investigations using fibrinogen coupled to colloidal gold (Fgn/Au) have shown that glycoprotein IIb-IIIa (GPIIb-IIIa) receptors are mobile and undergo centripetal reorganization on spread platelets following surface activation. The assembly of cytoplasmic actin and its constriction into an inner filamentous zone have been proposed as the mechanism driving the Fgn/Au receptor complexes across the plasma membrane. The present study has used cytochalasin B (CB), an agent known to inhibit actin assembly and cause breakdown of newly formed actin filaments to test that hypothesis. At a concentration which inhibited pseudopod formation and spreading, CB did not block movement of Fgn/Au receptor complexes toward platelet centers or into the open canalicular system (OCS). Channels of the OCS filled with Fgn/Au receptor complexes were evident in over 90% of the CB-treated, surface-activated platelets. The findings do not support the concept that assembly of cytoplasmic actin and its contraction move receptor-ligand complexes on the platelet plasma membrane.     

Localization of fibrinogen during ADP- or thrombin-induced aggregation of washed rabbit platelets

In contrast to human platelets, which aggregate poorly in response to ADP unless fibrinogen is present in the external medium, washed rabbit platelets form large aggregates in response to ADP without fibrinogen in the suspending medium. Addition of fibrinogen to the suspending medium of rabbit platelets frequently has little or no effect on the extent of ADP-induced platelet aggregation. We examined washed rabbit platelets by immunocytochemistry during ADP-induced aggregation and deaggregation and during thrombin-induced aggregation when the external medium did not contain added fibrinogen to determine if (a) fibrinogen was expressed on the surface of rabbit platelets that could support aggregation when the platelets were stimulated, or (b) fibrinogen secreted from the alpha granules supports platelet aggregation. Glutaraldehyde-fixed samples were prepared at different times after addition of ADP or thrombin, embedded in Lowicryl K4M, sectioned, incubated with sheep anti-rabbit fibrinogen, washed, reacted with gold-labeled anti-sheep IgG, and prepared for electron microscopy. The alpha granules of rabbit platelets were heavily labeled with immunogold; the platelet membrane was not labeled. During platelet aggregation and deaggregation in response to ADP, fibrinogen was not detectable on the platelet surface. In response to thrombin, large aggregates formed before fibrinogen was secreted from the alpha granules; fibrinogen was detectable focally at sites of granule discharge by 30-60 sec and fibrin formed by 3 min. Therefore, stimulated washed rabbit platelets can adhere to each other without large amounts of fibrinogen taking part in the close platelet-to-platelet contact, since aggregation occurs before detectable secretion, and large areas where the platelets are in contact are devoid of fibrinogen between the adherent membranes. Adhesion mechanisms not involving fibrinogen may support the aggregation of washed rabbit platelets.     

Redistribution of alpha-granules and their contents in thrombin- stimulated platelets

The redistribution of beta-thromboglobulin (beta TG), platelet Factor 4 (PF4), and fibrinogen from the alpha granules of the platelet after stimulation with thrombin was studied by morphologic and immunocytochemical techniques. The use of tannic acid stain and quick-freeze techniques revealed several thrombin-induced morphologic changes. First, the normally discoid platelet became rounder in form, with filopodia, and the granules clustered in its center. The granules then fused with one another and with elements of the surface-connected canalicular system (SCCS) to form large vacuoles in the center of the cell and near the periphery. Neither these vacuoles nor the alpha granules appeared to fuse with the plasma membrane, but the vacuoles were connected to the extracellular space by wide necks, presumably formed by enlargement of the narrow necks connecting the SCCS to the surface of the unstimulated cell. The presence of fibrinogen, beta TG, and PF4 in corresponding large intracellular vacuoles and along the platelet plasma membrane after thrombin stimulation was demonstrated by immunocytochemical techniques in saponin-permeabilized and nonpermeabilized platelets. Immunocytochemical labeling of the three proteins on frozen thin sections of thrombin-stimulated platelets confirmed these findings and showed that all three proteins reached the plasma membrane by the same pathway. We conclude that thrombin stimulation of platelets causes at least some of the fibrinogen, beta TG, and PF4 stored in their alpha granules to be redistributed to their plasma membranes by way of surface-connected vacuoles formed by fusion of the alpha granules with elements of the SCCS.     

Immunocytochemical evidence for the translocation of alpha-granule membrane glycoprotein IIb/IIIa (integrin alpha IIb beta 3) of human platelets to the surface membrane during the release reaction.

The localization of glycoprotein (GP) IIb/IIIa (integrin alpha IIb beta 3) in both resting and thrombin-activated platelets was studied immunocytochemically. By the preembedding method where only the GP IIb/IIIa molecules on the surface of platelets were immunostained, the distribution of protein A-colloidal gold label was randomly distributed along the surface membrane of resting platelets at a density of 18.0 +/- 2.7 gold particles/microns of membrane. At 15 s after stimulation by 0.1 U/ml of thrombin in an unstirred platelet suspension, the spheroid-shaped platelets with pseudopodia still had normal numbers of alpha-granules, and the density of gold particles was 19.7 +/- 3.6 particles/microns. At 5 min, the alpha-granules were no longer present because of the release reaction, and the density of gold particles significantly increased (27.0 +/- 3.7 particles/microns; p less than 0.01). In immuno-stained ultra-thin frozen sections, the gold particles were detected not only on the surface membrane, including the open canalicular system (OCS), but also on the alpha-granule membranes of resting platelets. At 30 s after thrombin stimulation the alpha-granules fused with the OCS, resulting in the formation of a swollen OCS, which still had gold particles on its membrane. At 5 min, the gold particles were detected on the membrane of the swollen OCS located near the surface membrane, while very few gold particles were present on the membrane of the OCS in the central part of the platelets. These results demonstrate that alpha-granule membrane GPIIb/IIIa translocates to the surface membrane through the membrane of the OCS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibrinogen distribution on surfaces and in organelles of ADP stimulated human blood platelets

The fibrinogen distribution in platelet organelles after ADP-stimulation was investigated with anti-human fibrinogen using protein A-gold applied to serial sections. Fibrinogen was detected in the so-called alpha-granules of platelets and also in granule protrusions which were observed after ADP-stimulation. The ends of these protrusions were formed as coated membranes and the tips were often in apposition to the surface connected membranes or the plasmalemma. At such places fusion events and hence signs of an exocytosis could be demonstrated by means of cryofixation and cryosubstitution. Examination of serial sections revealed fibrinogen on all these granule profiles. Surface connected membranes, free surfaces and the characteristic structure of the contact zones of aggregated platelets were also labelled by gold particles but less than anticipated. On the platelet surfaces and surface connected membranes fibrinogen was rarely demonstrable with ferritin-labelled anti-human fibrinogen on washed or thrombin-stimulated, almost fibrinogen free platelets. After addition of human fibrinogen to the thrombin stimulated and disaggregated platelets a part of the platelets aggregated spontaneously and formed characteristic contact zones. Anti-human fibrinogen was observed on the free surfaces, in filamentous bridges between the contact spaces and in a tubular surface connected membrane system with involvement of coated membranes at the central ends of these structures. The results indicate the following: all alpha-granules contain fibrinogen; after ADP-stimulation secretion takes place with involvement of coated membranes; during aggregation fibrinogen binds to platelet surfaces and forms contact spaces; fibrinogen is taken up by the surface connected system with involvement of coated membranes.     

Platelet membrane variations and their effects on δ-granule secretion kinetics and aggregation spreading among different species

Platelet exocytosis is regulated partially by the granular/cellular membrane lipids and proteins. Some platelets contain a membrane-bound tube, called an open canalicular system (OCS), which assists in granular release events and increases the membrane surface area for greater spreading. The OCS is not found in all species, and variations in membrane composition can cause changes in platelet secretion. Since platelet studies use various animal models, it is important to understand how platelets differ in both their composition and granular release to draw conclusions among various models. The relative phospholipid composition of the platelets with (mouse, rabbit) and without (cow) an OCS was quantified using UPLC-MS/MS. Cholesterol and protein composition was measured using an Amplex Red Assay and BCA Assay. TEM and dark field platelet images were gathered and analyzed with Image J. Granular release was monitored with single cell carbon fiber microelectrode amperometry. Cow platelets contained greater amounts of cholesterol and sphingomyelin. In addition, they yield greater serotonin release and longer δ granule secretion times. Finally, they showed greater spreading area with a greater range of spread. Platelets containing an OCS had more similarities in their membrane composition and secretion kinetics compared to cow platelets. However, cow platelets showed greater fusion pore stability which could be due to extra sphingomyelin and cholesterol, the primary components of lipid rafts. In addition, their greater stability may lead to many granules assisting in spreading. This study highlights fundamental membrane differences and their effects on platelet secretion.     

Endocytosis by human platelets: metabolic and freeze-fracture studies

The mechanism by which platelets endocytose or release particulate or soluble substances is poorly understood. Engulfed materials enter the open canalicular system (OCS) by a process akin to phagocytosis, but fusion of platelet granules with the OCS is rarely observed. Secretion of granule contents, a concomitant of the "release reaction" which occurs during platelet aggregation, does not take place by extrusion at the surface membrane as is true for other secretory cells. Some substances may be secreted without obvious granule loss. To examine whether structural properties of the platelet membrane could account for this unusual behavior, thin section and freeze-fracture analyses were performed on platelets which had undergone endocytosis under a variety of experimental conditions. After freeze-cleavage, most of the intramembranous particles (IMP) remain associated with the outer leaflet of the platelet plasma membrane. The sites where the OCS reaches the surface membrane are marked by pits on the cytoplasmic leaflet (P face) and by complementary protrusions on the outer leaflet (E face) of the membrane. Endocytosis of small particles and solutes takes place via these structures. This process is not energy dependent but arrested at 4 degrees C. Distension of the OCS does not appear to affect the size or number of the pits. On the other hand, large particles are taken up by membrane invagination without redistribution of IMP's and independent of the pits. This process is sensitive to metabolic inhibition. Thus, the studies have demonstrated the existence of two different pathways for platelet endocytosis which are postulated to be also involved in secretion. The selective release of substances contained in different granules may be related to the "inside-out" structure of the plasma and OCS membranes.     

Copper deficiency alters protein kinase C mediation of thrombin-induced dense granule secretion from rat platelets

Experiments were conducted to determine if copper deficiency enhances the rate of thrombin-induced dense granule secretion by modifying the major signal transduction pathways of rat platelets. Platelets were obtained from male, weanling Sprague-Dawley rats fed diets containing either deficient ( < 0.5 μg/g diet) or adequate (5.5 μg/g diet) copper for 5 weeks. Following stimulation with thrombin (0.1 U/mL), the rate of dense granule secretion as measured by ATP release was 160% higher in platelets from copper-deficient than from control rats. Inhibition of the rate of thrombin-induced ATP release by (6-aminohexyl)-1-naphthalene-sulfonamide, a calmodulin antagonist was independent of copper status. However, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, a protein kinase C inhibitor, inhibited the rate of ATP release only in platelets from copper-deficient rats. Aspirin had no effect on ATP release from platelets obtained from either copper-deficient or control rats. This suggests that copper deficiency alters the role of protein kinase C in regulating dense granule secretion. Analysis of autoradiographs showing [³²P]-labeled platelet proteins indicated that the phosphorylation of a 40 kDa protein, a known substrate for protein kinase C in platelets, was significantly less following thrombin stimulation in platelets from copper-deficient than from control rats. When protein kinase C was activated by phorbol 12-myristate 13-acetate prior to thrombin stimulation, ATP release was attenuated regardless of copper status. These findings suggest that protein kinase C can still function as a feedback inhibitor of platelet dense granule secretion in copper deficiency, but impaired activation of this enzyme following thrombin stimulation may prevent it from achieving full regulatory capacity.     

Role of platelet membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins

Treatment of platelets with thrombin was shown previously to induce rapid changes in tyrosine phosphorylation of several platelet proteins. In this report, we demonstrate that a variety of agonists which induce platelet aggregation also stimulate tyrosine phosphorylation of three proteins with apparent molecular masses of 84, 95, and 97 kD. Since platelet aggregation requires the agonist-induced activation of an integrin receptor (GP IIb-IIIa) as well as the binding of fibrinogen to this receptor, we examined the relationship between tyrosine phosphorylation and the function of GP IIb-IIIa. When platelets were examined under conditions that either precluded the activation of GP IIb-IIIa (prior disruption of the complex by EGTA at 37 degrees C) or the binding of fibrinogen (addition of RGDS or an inhibitory mAb), tyrosine phosphorylation of the 84-, 95-, and 97-kD proteins was not observed. However, although both GP IIb-IIIa activation and fibrinogen binding were necessary for tyrosine phosphorylation, they were not sufficient since phosphorylation was observed only under conditions in which the activated platelets were stirred and allowed to aggregate. In contrast, tyrosine phosphorylation was not dependent on another major platelet response, dense granule secretion. Furthermore, granule secretion did not require tyrosine phosphorylation of this set of proteins. These experiments demonstrate that agonist-induced tyrosine phosphorylation is linked to the process of GP IIb-IIIa-mediated platelet aggregation. Thus, tyrosine phosphorylation may be required for events associated with platelet aggregation or for events that follow aggregation.     

Differential role of protein kinase C delta isoform in agonist-induced dense granule secretion in human platelets

Several platelet agonists, including thrombin, collagen, and thromboxane A(2), cause dense granule release independently of thromboxane generation. Because protein kinase C (PKC) isoforms are implicated in platelet secretion, we investigated the role of individual PKC isoforms in platelet dense granule release. PKCdelta was phosphorylated in a time-dependent manner that coincided with dense granule release in response to protease-activated receptor-activating peptides SFLLRN and AYPGKF in human platelets. Only agonists that caused platelet dense granule secretion activated PKCdelta. SFLLRN- or AYPGKF-induced dense granule release and PKCdelta phosphorylation occurred at the same respective agonist concentration. Furthermore, AYPGKF and SFLLRN-induced dense granule release was blocked by rottlerin, a PKCdelta selective inhibitor. In contrast, convulxin-induced dense granule secretion was potentiated by rottlerin but was abolished by Go6976, a classical PKC isoform inhibitor. However, SFLLRN-induced dense granule release was unaffected in the presence of Go6976. Finally, rottlerin did not affect SFLLRN-induced platelet aggregation, even in the presence of dimethyl-BAPTA, indicating that PKCdelta has no role in platelet fibrinogen receptor activation. We conclude that PKCdelta and the classical PKC isoforms play a differential role in platelet dense granule release mediated by protease-activated receptors and glycoprotein VI. Furthermore, PKCdelta plays a positive role in protease-activated receptor-mediated dense granule secretion, whereas it functions as a negative regulator downstream of glycoprotein VI signaling.     

Adhesive ligand binding to integrin alpha IIb beta 3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK

Tyrosine phosphorylation of multiple platelet proteins is stimulated by thrombin and other agonists that cause platelet aggregation and secretion. The phosphorylation of a subset of these proteins, including a protein tyrosine kinase, pp125FAK, is dependent on the platelet aggregation that follows fibrinogen binding to integrin alpha IIb beta 3. In this report, we examined whether fibrinogen binding, per se, triggers a process of tyrosine phosphorylation in the absence of exogenous agonists. Binding of soluble fibrinogen was induced with Fab fragments of an anti-beta 3 antibody (anti-LIBS6) that directly exposes the fibrinogen binding site in alpha IIb beta3. Proteins of 50-68 KD and 140 kD became phosphorylated on tyrosine residues in a fibrinogen- dependent manner. This response did not require prostaglandin synthesis, an increase in cytosolic free calcium, platelet aggregation or granule secretion, nor was it associated with tyrosine phosphorylation of pp125FAK. Tyrosine phosphorylation of the 50-68-kD and 140-kD proteins was also observed when (a) fibrinogen binding was stimulated by agonists such as epinephrine, ADP, or thrombin instead of by anti-LIBS6; (b) fragment X, a dimeric plasmin-derived fragment of fibrinogen was used instead of fibrinogen; or (c) alpha IIb beta 3 complexes were cross-linked by antibodies, even in the absence of fibrinogen. In contrast, no tyrosine phosphorylation was observed when the ligand consisted of monomeric cell recognition peptides derived from fibrinogen (RGDS or gamma 400-411). Fibrinogen-dependent tyrosine phosphorylation was inhibited by cytochalasin D. These studies demonstrate that fibrinogen binding to alpha IIb beta 3 initiates a process of tyrosine phosphorylation that precedes platelet aggregation and the phosphorylation of pp125FAK. This reaction may depend on the oligomerization of integrin receptors and on the state of actin polymerization, organizational processes that may juxtapose tyrosine kinases with their substrates.     

Regulation of platelet dense granule secretion by the Ral GTPase-exocyst pathway

Non-hydrolyzable GTP analogues, such as guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp), induce granule secretion from permeabilized platelets in the absence of increased intracellular Ca(2+). Here, we show that the GppNHp-induced dense granule secretion from permeabilized platelets occurred concomitantly with the activation of small GTPase Ral. This secretion was inhibited by the addition of GTP-Ral-binding domain (RBD) of Sec5, which is a component of the exocyst complex known to function as a tethering factor at the plasma membrane for vesicles. We generated an antibody against Sec5-RBD, which abolished the interaction between GTP-Ral and the exocyst complex in vitro. The addition of this antibody inhibited the GppNHp-induced secretion. These data indicate that Ral mediates the GppNHp-induced dense granule secretion from permeabilized platelets through interaction with its effector, the exocyst complex. Furthermore, GppNHp enhanced the Ca(2+) sensitivity of dense granule secretion from permeabilized platelets, and this enhancement was inhibited by Sec5-RBD. In intact platelets, the association between Ral and the exocyst complex was induced by thrombin stimulation with a time course similar to that of dense granule secretion and Ral activation. Taken together, our results suggest that the Ral-exocyst pathway participates in the regulation of platelet dense granule secretion by enhancing the Ca(2+) sensitivity of the secretion.     

Regulation of the phosphoinositide hydrolysis pathway in thrombin-stimulated platelets by a pertussis toxin-sensitive guanine nucleotide-binding protein. Evaluation of its contribution to platelet activation and comparisons with the adenylate cyclase inhibitory protein, Gi

In platelets activated by thrombin, the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C produces inositol 1,4,5-triphosphate (IP3) and diacylglycerol, metabolites which are known to cause Ca2+ release from the platelet dense tubular system and granule secretion. Previous studies suggest that phospholipase C activation is coupled to platelet thrombin receptors by a guanine nucleotide-binding protein or G protein. The present studies examine the contribution of this protein to thrombin-induced platelet activation and compare its properties with those of Gi, the G protein which mediates inhibition of adenylate cyclase by thrombin. In platelets permeabilized with saponin, nonhydrolyzable GTP analogs reproduced the effects of thrombin by causing diacylglycerol formation, Ca2+ release from the dense tubular system and serotonin secretion. In intact platelets, fluoride, which by-passes the thrombin receptor and directly activates G proteins, caused phosphoinositide hydrolysis and secretion. Fluoride also caused an increase in the platelet cytosolic free Ca2+ concentration that appeared to be due to a combination of Ca2+ release from the dense tubular system and increased Ca2+ influx across the platelet plasma membrane. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits G protein function, inhibited the ability of thrombin to cause IP3 and diacylglycerol formation, granule secretion, and Ca2+ release from the dense tubular system in saponin-treated platelets. Increasing the thrombin concentration overcame the effects of GDP beta S on secretion without restoring diacylglycerol formation. The effects of GDP beta S on platelet responses to thrombin which had been subjected to partial proteolysis (gamma-thrombin) were similar to those obtained with native alpha-thrombin despite the fact that gamma-thrombin is a less potent inhibitor of adenylate cyclase than is alpha-thrombin. Thrombin-induced diacylglycerol formation and 45Ca release were also inhibited when the saponin-treated platelets were preincubated with pertussis toxin, an event that was associated with the ADP-ribosylation of a protein with Mr = 41.7 kDa. At each concentration tested, the inhibition of thrombin-induced diacylglycerol formation by pertussis toxin paralleled the inhibition of thrombin's ability to suppress PGI2-stimulated cAMP formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fibrinogen Manchester. Detection of a heterozygous phenotype in the intraplatelet pool.

Family members heterozygous for the congenitally abnormal fibrinogen designated fibrinogen Manchester, A alpha 16Arg----His, have previously been shown by h.p.l.c. and amino acid analysis to release a variant fibrinopeptide, [His16]fibrinopeptide A, from plasma fibrinogen after the addition of thrombin. The present study was designed to determine if the same abnormal phenotype was also present in the intraplatelet fibrinogen pool. Fresh platelets were washed in buffers containing EDTA until it could be shown that all washable plasma fibrinogen was removed. Normal platelets were then lysed by freezing and thawing to release their intracellular proteins, which were then treated with thrombin. The fibrinopeptides, cleaved from the intraplatelet fibrinogen, could be detected by an optimized h.p.l.c. technique. Quantification of the intraplatelet fibrinogen gave a result (means +/- S.D., n = 5) of 110 +/- 30 and 90 +/- 30 micrograms/10(9) platelets, when determined by h.p.l.c. quantification of fibrinopeptide B content and fibrinogen fragment E radioimmunoassay respectively. Examination of fibrinopeptides released from the platelet fibrinogen from the family with fibrinogen Manchester with the same techniques showed elution peaks in the same positions as both [His16]fibrinopeptide A and normal fibrinopeptide A. The identity of these peaks was further substantiated by analysis of the h.p.l.c. peaks by using specific radioimmunoassay to fibrinopeptide A. Our results therefore demonstrate that platelet fibrinogen expresses the heterozygous A alpha 16His phenotype. This supports the view that the A alpha chains of platelet and plasma fibrinogen are produced from a single genetic locus.     

Inhibitors of Na+/H+ exchange block stimulus-provoked arachidonic acid release in human platelets. Selective effects on platelet activation by epinephrine, ADP, and lower concentrations of thrombin

We have observed previously that removal of extraplatelet Na+ blocks platelet secretion of dense granule contents in response to epinephrine, ADP, and 0.004 unit/ml thrombin, all agents which must mobilize arachidonic acid for its subsequent conversion to cyclooxygenase products in order to elicit platelet secretion. The present studies demonstrate that removal of extraplatelet Na+ blocks arachidonic acid mobilization in response to epinephrine, ADP, and 0.004 unit/ml thrombin without altering arachidonic acid conversion to thromboxane A2. The data also provide several lines of evidence which suggest that the blockade of arachidonic acid release due to removal of extraplatelet Na+ is a manifestation of blockade of Na+/H+ exchange system. 1) There is a concentration-dependent effect of extraplatelet Na+ (EC50 congruent to 55 mM) on [3H]arachidonic acid release such that mobilization is observed when [Na+]o greater than [Na+]i. 2) Increasing extraplatelet [H+] (i.e. decreasing extraplatelet pH from pH 7.35 to 6.8) causes a concentration-dependent decline in stimulus-provoked [3H]arachidonic acid release. 3) Ethylisopropylamiloride and other potent 5-amino analogs of amiloride block [3H]arachidonic acid release with a potency that parallels their effects on Na+/H+ exchange in other cellular systems. None of the above manipulations alter primary aggregation induced by epinephrine, ADP, or 0.004 unit/ml thrombin, indicating that stimulus-receptor binding, subsequent exposure of fibrinogen receptors, and fibrinogen-mediated platelet-platelet cross-linking are not significantly inhibited by [3H]arachidonic acid release in response to greater than 0.1 unit/ml thrombin, a stimulus that can elicit platelet secretion in the absence of products of the cyclooxygenase pathway. Therefore, Na+/H+ exchange may selectively modulate arachidonic acid mobilization in response to the so-called "weak agonists," agonists that require this mobilization to effect vigorous platelet aggregation and dense granule secretion.     

Thrombocytin, a serine protease from Bothrops atrox venom. 2. Interaction with platelets and plasma-clotting factors.

Thrombocytin, a serine protease from Bothrops atrox venom, caused platelet aggregation and release of platelet constituents at a concentration of 10(-7) M and clot retraction at a concentration of 2 x 10(-9) M. Thrombocytin was slightly more active when tested on platelets in plasma than on washed platelets suspended in Tyrode--albumin solution. Thrombin was 5 times more active than thrombocytin when tested on platelets in plasma and 50 times more active when tested on washed platelets. The patterns or release induced by thrombocytin and thrombin were similar. Prostaglandin E1 (10(-5) M) produced complete inhibition of platelet release induced by thrombocytin and thrombin. Indomethacin (10(-4) M) was without any effect. Antithrombin III, in the presence of heparin, inhibited the action of thrombocytin on platelets and on a synthetic peptide substrate (Tos-Gly-Pro-Arg-pNA.HCl). formation of an antithrombin III--thrombocytin complex was demonstrated on NaDodSO4--polyacrylamide gel electrophoresis. Hirudin and alpha 1-antitrypsin did not inactivate thrombocytin. Thrombocytin had a low fibrinogen-clotting activity (less than 0.06% that of thrombin). Thrombocytin also caused progressive degradation of the alpha chain of human fibrinogen, and it cleaved prothrombin, releasing products similar to intermediate 1 and fragment 1 produced by thrombin. Thrombocytin activated factor XIII by limited proteolysis and increased the procoagulant activity of factor VIII in a manner analogous to that of thrombin.     

Specific binding of blood coagulation factor XIIIa to thrombin-stimulated platelets

We studied the binding of 125I-platelet and plasma Factor XIII (125I-Factor XIII) to human platelets. When 125I-Factor XIII was incubated with gel-filtered platelets, calcium chloride (5 mM) and thrombin (1 unit/ml) at 37 degrees C, saturable binding was observed. Half-maximal binding occurred at 1 min. Binding was inhibited 93% by a 100-fold molar excess of unlabeled ligand but not by other purified proteins. Greater than 87% of platelet-bound radioactivity migrated as thrombin-cleaved a-chains (a'-chains) in sodium dodecyl sulfate-polyacrylamide gels indicating that Factor XIIIa but not Factor XIII binds to platelets. 125I-Factor XIIIa does not bind to unstimulated platelets. When platelet secretion was blocked, binding was markedly inhibited. 125I-Factor XIIIa bound minimally to platelets stimulated with agonists other than thrombin. Thus, binding is dependent on platelet activation, as well as modification of platelets by thrombin. 125I-Factor XIIIa bound to gamma-thrombin-stimulated platelets, at concentrations which did not clot fibrinogen. Therefore, Factor XIIIa is not bound to fibrin associated with platelets. Binding was only partially reversible. Approximately 12,000 molecules of Factor XIIIa were bound per platelet. 125I-Factor XIIIa bound normally to platelets from patients with severe Glanzmann's thrombasthenia indicating that 125I-Factor XIIIa does not bind to platelet glycoproteins IIb or IIIa, or platelet-bound fibrinogen. Chymotrypsin treatment of platelets inhibited 125I-Factor XIIIa binding by 78% without inhibiting secretion. Methylamine and putrescine, Factor XIIIa substrates, and N-ethylmaleimide, an active site inhibitor, did not inhibit binding. Factor XIIIa bound to platelets was enzymatically active and catalyzed [3H]putrescine incorporation into platelet proteins. The specific binding of Factor XIIIa to platelets suggests it may play a role in physiologic reactions involving platelets.     

Distinct but critical roles for integrin alphaIIbbeta3 in platelet lamellipodia formation on fibrinogen, collagen-related peptide and thrombin

Integrins are the major receptor type known to facilitate cell adhesion and lamellipodia formation on extracellular matrix proteins. However, collagen-related peptide and thrombin have recently been shown to mediate platelet lamellipodia formation when presented as immobilized surfaces. The aims of this study were to establish if there exists a role for the platelet integrin alpha(IIb)beta(3) in this response; and if so, whether signalling from the integrin is required for lamellipodia formation on these surfaces. Real-time analysis was used to compare platelet morphological changes on surfaces of fibrinogen, collagen-related peptide or thrombin in the presence of various pharmacological inhibitors and platelets from 'knockout' mice. We demonstrate that collagen-related peptide and thrombin stimulate distinct patterns of platelet lamellipodia formation and elevation of intracellular Ca(2+) to that induced by the integrin alpha(IIb)beta(3) ligand, fibrinogen. Nevertheless, lamellipodia formation on collagen-related peptide and thrombin is dependent upon engagement of alpha(IIb)beta(3), consistent with release of alpha(IIb)beta(3) ligand(s) from platelet granules. However, the requirement for signalling by the integrin on fibrinogen can be bypassed by the addition of thrombin to the solution. These observations reveal a critical role for alpha(IIb)beta(3) in forming lamellipodia on collagen-related peptide and thrombin which is dependent on its ability to function as an adhesive receptor but not necessarily on its ability to signal. These results suggest that integrins may play an important role in lamellipodia formation triggered by nonintegrin ligands in platelets and possibly in other cell types.     

Involvement of platelet glycoprotein IIb-IIIa (alpha IIb-beta 3 integrin) in thrombin-induced synthesis of phosphatidylinositol 3',4'-bisphosphate.

32P-Labeled human platelets were incubated with thrombin (1 unit/ml) for 5 min at 37 degrees C under conditions allowing maximal synthesis of [32P]phosphatidylinositol 3',4'-bisphosphate (PtdIns(3,4)P2). Incorporation of 32P into the latter phosphoinositide was dose-dependently reduced (to a maximal level averaging 60%) by the tetrapeptide RGDS, an inhibitor of fibrinogen binding to activated glycoprotein IIb-IIIa (alpha IIb-beta 3 integrin). Identical results were obtained with the fibrinogen gamma-chain dodecapeptide HHLGGAKQAGDV, whereas the tripeptide RGD and the tetrapeptide RGES displayed reduced or undetectable effects on 32P labeling of PtdIns(3,4)P2, respectively, in good correlation with their ability to inhibit platelet aggregation and fibrinogen binding to activated alpha IIb-beta 3 integrin. In addition, pathological platelets from three patients suffering thrombasthenia, which lack alpha IIb-beta 3 integrin and fail to aggregate in response to thrombin, displayed hardly detectable increases in the 32P labeling of PtdIns(3,4)P2. In contrast, thrombin-stimulated synthesis of PtdIns(3,4)P2 was unaltered in other deficient platelets lacking the glycoprotein Ib-IX complex (Bernard-Soulier syndrome). Although additional pathways seem to be involved in the regulation of phosphatidylinositol-3-kinase, these data indicate a strong relationship between platelet aggregation involving fibrinogen binding to activated alpha IIb-beta 3 integrin and the synthesis of the novel phosphoinositides phosphorylated at position D-3 of the inositol ring.