Thrombin binding to platelets defines functional receptors: inhibition of thrombin-induced platelet activation by catalytically-inactivated thrombin

It has been widely questioned as to whether the observed binding of a-thrombin to intact platelets defines receptors coupled to signal transduction or merely thrombin binding sites. We have now shown that at α-thrombin concentrations sufficient to induce a full shape change response without aggregation (0.1 nM), PPACK-thrombin (that is, α-thrombin treated with the irreversible active site inhibitor D-phenylalanyl-L-prolyl-L-arginine chloromethylketone) dose-dependently inhibits platelet shape change (IC(50)～70 nM), the concomitant increases in [Ca(2+)Ii (IC(50)～75 nM) and ATP secretion (IC(50)～50 nM). Since PPACK-thrombin competes fully in the binding of a-thrombin to high, moderate and low affinity sites on intact platelets, these results show that this binding defines functional receptors coupled to platelet activation.     

Characterization of Binding of an RGD Mimetic, [3H]-SC-52012, to Platelet GPIIb/IIIa

In order to compare binding of small peptide mimetics on activated vs resting platelets and with fibrinogen (fgn) on activated platelets, the binding of [³H]-SC-52012, a low molecular weight (483) mimetic of the RGDF sequence present in fgn, was evaluated. This compound is a potent inhibitor of fgn binding to activated platelets, IC, 9.0 ± 0.6 nM (mean ± SEM), and inhibits ADP induced human platelet aggregation (IC, 44 ± 5 nM). The dissociation constant (Kd) of [³H]-SC-52012 was 21.6 ± 4.7 nM (n = 13) in ADP-induced human washed platelets while the Kd for resting platelets was 156 ± 8.3 nM (n = 3). The maximum number of binding sites on ADP-activated and resting platelets were 60846 ± 7158 and 59464 ± 5898 molecules/platelet, respectively. By comparison, results with [¹²⁵I]-fgn binding to activated platelets gave values of 363 ± 73 nM and 58046 ± 6386 molecules/platelet (n = 8) for the Kd and receptor number, respectively. These data suggest that the small molecule binds regardless of activation state of the platelet with only a change in affinity. [³H]-SC-52012 could be displaced by unlabelled SC-52012 with an IC₅₀ of 135 ± 20 nM.     

Differential activation and inhibition of human platelet thrombin receptors by structurally distinct α-, β- and γ-thrombin

The development of drugs to neutralize the action of thrombin has to date focused on the α form of the protease. It is generally agreed that inactive prothrombin is proteolytically converted to active α-thrombin which may be further hydrolyzed to β- and γ-thrombin. While all three forms of the enzyme retain catalytic activities, only α-thrombin is presumed to be physiologically important. The β- and γ-thrombin are presumed to be degradation products of no physiological significance. Our demonstration that β- and γ-thrombin selectively activate PAR-4 in this and a previous report (J. Biol. Chem. 276, 21173–21183, 2001) necessitates a reevaluation of how we view their physiological roles and how we approach the pharmacological regulation of their actions. β-Thrombin, like γ-thrombin, at nM levels selectively activates PAR-4. This was demonstrated by full retention of aggregatory activity with platelets whose PAR-1 and GP Ib receptors were inactivated. Furthermore, the β-thrombin response was abrogated by desensitizing platelets with suboptimal levels of the thrombin receptor activating peptide for PAR-4 (TRAP-4). For β-thrombin and γ-thrombin to have a physiological role, it is necessary to show they can be generated under physiological conditions. We demonstrate, for the first time, that α-thrombin is hydrolyzed in less than 1?min by activated factor X at physiological pH, in vitro. This implies that α-thrombin may be rapidly converted to β-thrombin and/or γ-thrombin in vivo in the proper microenvironment. The differential activation of the three platelet thrombin receptors by α-, β- and γ-thrombin implies selective structural variations between these thrombin species. Structural differences are likely to account for the marked differential responses observed with the antithrombotic, hirudin, which inhibits α-thrombin, is a slightly weaker inhibitor of β- thrombin and a very weak inhibitor of γ-thrombin-induced platelet aggregations. The converse order of inhibition is observed with the physiological protease inhibitor, α₁-antitrypsin. Finally, a non-traditional inhibitor, histone-1, selectively inhibits only β- and γ-thrombin, primarily at the receptor level of PAR-4 rather than on the thrombin molecule. Trypsin, like β- and γ-thrombin, activates PAR-4 and is also inactive with TRAP-4 desensitized platelets. Therefore, it was reasoned that trypsin would be more structurally similar to γ-thrombin than to α-thrombin. The analysis of the crystalline structures of α-, γ-thrombin and trypsin from the databases confirm that this is the case. These findings should help to elucidate structure-function relationships of the different thrombins and may aid in the development of new anti-thrombotic drugs.     

Thrombin binding to platelets and their activation in plasma

Summary. The interactions of α-thrombin with platelets are critical in haemostasis and arterial thrombosis. This study established methods for characterizing the binding of α-thrombin to platelets and some of its consequences in platelet-rich plasma. The binding of α-thrombin to platelets and the subsequent platelet activation were quantified by flow cytometry, using affinity purified polyclonal antibodies to human α-thrombin and a monoclonal antibody to GMP-140, respectively. Dose-dependent binding of α-thrombin to platelets and their activation occurred in parallel, both reaching the maxima for each enzyme concentration within 10 s after → 1.0 n m α-thrombin was added to recalcified PRP containing 1 μ m recombinant tick anticoagulant peptide. The tick anticoagulant peptide abrogated prothrombin activation in the platelet-rich plasma. α-Thrombin binding to platelets, and their activation, were abrogated by a monoclonal antibody to the hirudin tail-like domain of the seven transmembrane thrombin receptor on platelets. Therefore this receptor represents an important site for α-thrombin binding to platelets suspended in plasma. d -Phe-Pro-ArgCH₂-α-thrombin only bound to platelets when its concentration was → 100 n m , and it did so without inhibiting platelet activation by α-thrombin. Whereas concentrations of hirudin equimolar to those of α-thrombin failed to abrogate α-thrombin-mediated activation of platelets, a 10-fold molar excesses of hirudin over α-thrombin abrogated α-thrombin binding to platelets. The demonstration that → 1.0 n m α-thrombin can bind to platelets and initiate their activation raises the possibility that the levels of thrombin generated in venous and arterial thrombosis contribute to platelet activation in vivo .     

PPACK-thrombin inhibits thrombin-induced platelet aggregation and cytoplasmic acidification but does not inhibit platelet shape change

We have re-evaluated the previously reported ability of TLCK-thrombin (N alpha-tosyl-L-lysine chloromethyl ketone-treated alpha-thrombin) and PPACK-thrombin (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-treated alpha-thrombin) to inhibit alpha-thrombin-induced platelet activation (Harmon JT, Jamieson GA: J Biol Chem 261:15928, 1986; and Harmon JT, Jamieson GA: Biochemistry 27:2151, 1988). Despite several cycles of derivatization with TLCK (10,000-fold molar excess), preparations of TLCK-thrombin have been found to contain about 4% residual alpha-thrombin activity, suggesting that these preparations are an equilibrium mixture of TLCK-thrombin and alpha-thrombin and cannot be used for evaluating competition between these two agents. In contrast, alpha-thrombin activity was completely inhibited by PPACK at 15-fold molar excess. PPACK-thrombin, free of unreacted PPACK and devoid of residual alpha-thrombin activity, did not markedly affect platelet shape change at concentrations as high as 1 mumol/L, but inhibited aggregation and secretion in intact platelets activated with the minimal concentration of alpha-thrombin causing a full response (0.3 to 0.5 nmol/L) and yielded a 50% inhibition constant (IC50) for inhibition of aggregation by PPACK-thrombin of 110 nmol/L. This inhibition was specific for alpha-thrombin-induced platelet activation, and no inhibition was seen with activation induced by ADP, collagen, epinephrine, ristocetin, or arachidonate. At these low alpha-thrombin concentrations (approximately 0.4 nmol/L), a persistent cytoplasmic acidification was observed of -0.062 +/- 0.016 pH units, although alkalinization was observed at higher alpha-thrombin concentrations (greater than 1 nmol/L). While inhibition of aggregation and secretion occurred when alpha-thrombin and PPACK-thrombin were added simultaneously, inhibition of cytoplasmic acidification and of the elevation of cytoplasmic [Ca2+] induced by low concentrations of alpha-thrombin (0.4 nmol/L) occurred only if platelets were preincubated with PPACK-thrombin for 5 minutes before the addition of alpha-thrombin. In platelets treated with Serratia marcescens protease to remove glycoprotein lb (GPlb), alpha-thrombin-induced shape change was attenuated but persisted in the presence of a high concentration (2 mumol/L) of PPACK-thrombin, although aggregation and secretion were inhibited, as seen in intact platelets. The IC50 value for inhibition of aggregation by PPACK-thrombin was approximately 1 mumol/L at the higher alpha-thrombin concentrations (5 nmol/L) required for full activation in this case. These results suggest that PPACK-thrombin may be a useful probe of platelet function since it specifically blocks platelet aggregation and secretion induced by alpha-thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)     

The P2Y1 receptor, necessary but not sufficient to support full ADP-induced platelet aggregation, is not the target of the drug clopidogrel

Recently we showed that the P2Y₁ receptor coupled to calcium mobilization is necessary to initiate ADP-induced human platelet aggregation. Since the thienopyridine compound clopidogrel specifically inhibits ADP-induced platelet aggregation, it was of interest to determine whether the P2Y₁ receptor was the target of this drug. Therefore we studied the effects of clopidogrel and of the two specific P2Y₁ antagonists A2P5P and A3P5P on ADP-induced platelet events in rats. Although clopidogrel treatment (50 mg/kg) greatly reduced platelet aggregation in response to ADP as compared to untreated platelets, some residual aggregation was still detectable. In contrast, A2P5P and A3P5P totally abolished ADP-induced shape change and aggregation in platelets from both control and clopidogrel-treated rats. A2P5P and A3P5P (100 μM ) totally inhibited the [Ca²⁺]_i rise induced by ADP (0.1 μM ) in control and clopidogrel-treated platelets, whereas clopidogrel treatment had no effect. Conversely, the inhibition of adenylyl cyclase induced by ADP (5 μM ) was completely blocked by clopidogrel but not modified by A2P5P or A3P5P (100 μM ). A3P5P (1 m M ) reduced the number of [³³P]2MeSADP binding sites on control rat platelets from 907 ± 50 to 611 ± 25 per platelet. After clopidogrel treatment, binding of [³³P]2MeSADP decreased to 505 ± 68 sites per platelet and further decreased to 55 ± 12 sites in the presence of A3P5P (1 m M ). In summary, these results demonstrate that the platelet P2Y₁ receptor responsible for the initiation of aggregation in response to ADP is not the target of clopidogrel. Platelets may express another, as yet unidentified, P2Y receptor, specifically coupled to the inhibition of adenylyl cyclase and necessary to induce full platelet aggregation, which could be the target of this drug.     

Specific 125I Neuropeptide Y Binding to Intact Cultured Bovine Adrenal Medulla Capillary Endothelial Cells

Objective: This study involved the pharmacological detection and characterization of binding sites for the neuromodulator neuropeptide Y (NPY) in an in vitro preparation of capillary endothelial cells derived from bovine adrenal medulla. Methods: Equilibrium binding assays were conducted on intact cells with ¹²⁵I Bolton-Hunter labeled NPY (¹²⁵I-BH-NPY). The specificity of the high-affinity binding site was evaluated in competition experiments with cold NPY, (Leu³¹, Pro³⁴)NPY (a Y₁ receptor ligand, Y₁RL), NPY_13–36 (a Y₂ receptor ligand, Y₂RL), and two other members of the pancreatic polypeptide-fold (PP-fold) family: peptide YY (PYY) and avian pancreatic polypeptide (APP). Forskolin-stimulated adenylate cyclase activity was assessed to detect the participation of this second messenger pathway in the neuromodulator action at the studied cell preparation. Results: Nonlinear regression analysis of the binding data indicated the existence of high-affinity binding sites with an equilibrium dissociation constant (K_d) value of 39.00 ± 12.84 nM and a maximal binding (B_max) of 489.89 ± 155.49 fmol/10⁶ cells (mean ± SE, n = 6). NPY, Y₁RL, and PYY displayed a concentration that inhibits the specific binding by 50% IC₅₀ (nM) values of 4.06 ± 1.66 (n = 4), 2.94 ± 0.75 (n = 5), and 18.36 ± 10.36 (n = 3), respectively. APP and Y₂RL were unable to compete with ¹²⁵I-NPY in the concentration range 0.001–1 μM. Further evaluation of second messenger pathways suggested that NPY binding sites in this model are coupled to the inhibition of adenylate cyclase. NPY significantly inhibited the forskolin-stimulated adenosine cyclic 3′,5′-(hydrogen phosphate) (cAMP) accumulation with a maximal effect of 37.03 ± 6.28%, n = 5 and an IC₅₀ of 5.96 ± 1.87 nM. The Y₁RL produced a comparable response (IC₅₀ = 5.35 ± 1.39 nM, n = 4; maximal inhibition of 61.05 ± 13.03%) and Y₂LR had no detectable effect at a similar concentration range. Conclusions: The results demonstrate the existence of a Y₁ receptor in the adrenal medulla capillary endothelial cells, which may be relevant to the postjunctional effect of NPY on this gland.     

Demonstration of a novel circulating anti-prostacyclin receptor antibody

Although coronary artery disease (CAD) is appreciated to be accelerated in patients with chronic spinal cord injury (SCI), the underlying mechanism of CAD in SCI remains obscure. We have recently shown that platelets from subjects with SCI develop resistance to the inhibitory effect of prostacyclin (PGI₂) on the platelet stimulation of thrombin generation. The loss of the inhibitory effect was due to the loss of high-affinity prostanoid receptors, which may contribute to atherogenesis in SCI. Incubation of normal, non-SCI platelets in SCI plasma (n = 12) also resulted in the loss of high-affinity binding of PGI₂ (Kd₁ = 9.1 ± 2.0 nM; n₁ = 170 ± 32 sites per cell vs. Kd₁ = 7.2 ± 1.1 nM; n₁ = 23 ± 8 sites per cell), with no significant change in the low-affinity receptors (Kd₂ = 1.9 ± 0.1 μM; n₂ = 1,832 ± 232 sites per cell vs. Kd₂ = 1.6 ± 0.1 μM; n₂ = 1,740 ± 161 sites per cell) as determined by Scatchard analysis of the binding of [³H]PGE₁. The loss of high-affinity PGI₂ binding led to the failure of PGI₂ to inhibit the platelet-stimulated thrombin generation. The increase of cellular cyclic AMP level, mediated through the binding of PGI₂ to low-affinity receptors in platelets, was unaffected in SCI platelets. PAGE and immunoblot of SCI plasma showed the presence of an IgG band, which specifically blocked the binding of [³H]PGE₁ to the high-affinity PGI₂ receptors of normal platelets. PAGE of the reduced IgG band, the amino acid sequence of the novel band as a heavy chain of IgG that inhibits the binding of [³H]PGE₁ to the high-affinity platelet PGI₂ receptor, demonstrates that the specific recognition and inhibition of high-affinity PGI₂ binding to platelets was due to an anti-prostacyclin receptor antibody present in SCI plasma.     

Identification of the active region responsible for the anti-thrombotic activity of anopheline anti-platelet protein from a malaria vector mosquito

We previously identified an anti-platelet protein, anopheline anti-platelet protein (AAPP), from the salivary gland of female Anopheles stephensi (a mosquito vector of human malaria). AAPP specifically blocks platelet adhesion to collagen by binding directly to collagen and subsequently causing platelet aggregation. The aim of this study was to identify the active region of AAPP responsible for the anti-thrombotic activity because we hypothesized that AAPP could be used as a candidate anti-platelet drug. Various truncated forms of AAPP were produced using an Escherichia coli expression system. Each protein was examined for binding activities to soluble/fibrillar collagen and anti-thrombotic activity using a plate assay and platelet/whole blood aggregation study, respectively. Among the truncated forms examined, only a protein encoded by exon 3–4 (rAAPPex_3–4) effectively bound to soluble/fibrillar collagen in a concentration-dependent and saturable manner. The EC₅₀ values of full-length AAPP and rAAPPex_3–4 for soluble collagen binding were 35?nM and 36?nM, respectively. In contrast to soluble collagen, there was a difference in binding affinity to fibrillar collagen between full-length AAPP and rAAPPex_3–4, with EC₅₀ values of 31?nM and 51?nM, respectively. rAAPPex_3–4 also inhibited aggregation of platelets/whole blood, and the IC₅₀ values of full-length AAPP and rAAPPex_3–4 for platelet aggregation were 35?nM and 93?nM, respectively. These results indicated that the essential moiety of AAPP for collagen binding and anti-thrombotic activity was in the region encoded by exon 3–4, which is highly conserved among the counterpart regions of other mosquito species.     

Binding of Thrombin to Human Platelets and its Possible Significance

SUMMARY Thrombin binds tightly to human platelets. The binding reaction is dependent on the thrombin concentration used. At a physiologically significant thrombin concentration, there are about 500 binding sites per platelet with an apparent dissociation constant of 0.02 u/ml. Autoradiography studies of platelets treated with labelled thrombin showed that the thrombin was located on the platelet surface. Separation of the subcellular fractions of platelets treated with labelled thrombin by density gradient centrifugation revealed that the membrane area contained over 80% of the radioactivity initially applied. Furthermore, isolated platelet membranes bind thrombin similar to intact platelets. These data suggest that the receptors for thrombin are located on the platelet membrane. Cytochalasin A, cytochalasin B or prostaglandin E₁ did not have any effect on thrombin binding although these agents inhibited platelet aggregation. Thus, binding of thrombin is not sufficient for aggregation of platelets and other steps are involved. These agents do not affect the induction of stimulation but interfere at a later step in the thrombin-platelet interaction. On the other hand, hirudin completely inhibited binding of thrombin to platelets. It appears that the platelet receptor recognizes that part of the thrombin molecule on its surface which is blocked by hirudin. Binding studies with serotonin loaded platelets showed a close correlation between thrombin binding and the release reaction.     

Influence of α-Methyldopa Treatment on Adrenergic Receptor Binding in Rat Forebrain

We studied the influence of 6 days treatment with α₃-methyldopa (50 mg/kg s.c.) twice daily on radioligand binding to α₁ ((³H)prazosin), α₂ ((³H)clonidine) and β((³H)dihydroalprenolol (D₃HA)) receptors in rat forebrain. A 27% rise (p lt; 0.05) in the B_max for (³H)prazosin without change in Kd was found. Following α-methyldopa, the K_d for (³H)clonidine was increased from 1.78 ± 19 to 3.03 ± 0.30 nM and B_max fell from 197 ± 20 to 167 ± 19 fmoles/mg protein (p < 0.05). These findings suggest that chronic α-methyldopa therapy induces changes in α₁ and α₂ receptors which may modify the antihypertensive effect of α-methyldopa.     

Lysophosphatidic acid-induced platelet shape change revealed through LPA1–5 receptor-selective probes and albumin

Lysophosphatidic acid (LPA), a component of mildly-oxidized LDL and the lipid rich core of atherosclerotic plaques, elicits platelet activation. LPA is the ligand of G protein-coupled receptors (GPCR) of the EDG family (LPA_1–3) and the newly identified LPA_4–7 subcluster. LPA₄, LPA₅ and LPA₇ increase cellular cAMP levels that would induce platelet inhibition rather than activation. In the present study we quantified the mRNA levels of the LPA_1–7 GPCR in human platelets and found a rank order LPA₄ = LPA₅ > LPA₇ > LPA₆ = LPA₂ ? LPA₁ > LPA₃. We examined platelet shape change using a panel of LPA receptor subtype-selective agonists and antagonists and compared them with their pharmacological profiles obtained in heterologous LPA_1–5 receptor expression systems. Responses to different natural acyl and alkyl species of LPA, and octyl phosphatidic acid analogs, alpha-substituted phosphonate analogs, N-palmitoyl-tyrosine phosphoric acid, N-palmitoyl-serine phosphoric acid were tested. All of these compounds elicited platelet activation and also inhibited LPA-induced platelet shape change after pre-incubation, suggesting that receptor desensitization is likely responsible for the inhibition of this response. Fatty acid free albumin (10 µM) lacking platelet activity completely inhibited platelet shape change induced by LPA with an IC₅₀ of 1.1 µM but had no effect on the activation of LPA_1,2,3,&5 expressed in endogenously non-LPA-responsive RH7777 cells. However, albumin reduced LPA₄ activation and shifted the dose-response curve to the right. LPA₅ transiently expressed in RH7777 cells showed preference to alkyl-LPA over acyl-LPA that is similar to that in platelets. LPA did not increase cAMP levels in platelets. In conclusion, our results with the pharmacological compounds and albumin demonstrate that LPA does not induce platelet shape change simply through activation of LPA_1–5, and the receptor(s) mediating LPA-induced platelet activation remains elusive.     

Effect of ADP-induced Shape Change on Incorporation of 32P into Platelet Phosphatidic Acid and Mono-, Di- and Triphosphatidyl Inositol

Summary . Experiments were carried out to determine whether the ADP-induced increase in ³²P-content of phosphatidic acid (PA), monophosphatidylinositol (MPI), diphosphatidylinositol (DPI) or triphosphatidylinositol (TPI) of washed ³²PO₄ labelled platelets is related to platelet aggregation or to the change in shape induced by ADP. Washed platelets were labelled with ³²PO₄, washed, and resuspended in medium containing unlabelled PO₄. With rabbit platelets, either the omission of Ca⁺⁺ from the suspending medium or the addition of ethyleneglycoltetraacetic acid (EGTA) in amounts sufficient to chelate Ca ⁺⁺ in the suspending medium, prevented ADP-induced platelet aggregation. Lack of free Ca⁺⁺ in the suspending medium did not prevent the ADP-induced shape change or the increased labelling of PA and DPI. In suspensions containing EGTA, the increase in ³²P-content of PA induced by varying amounts of ADP was compared to the extent of the shape change. ADP in a final concentration of 10^?6m or greater, produced both the maximum increase in ³²P-content of PA and the maximum degree of shape change. Washed platelets from pigs or humans without fibrinogen in the suspending medium showed a shape change following the addition of ADP but no aggregation, whereas in suspensions containing fibrinogen, the platelets showed both a shape change and aggregation. In suspension with and without fibrinogen, the ADP induced increase in ³²P-content of PA in human platelets and of PA+ MPI and DPI in pig platelets was the same. Prostaglandin E₁ (PGE₁), 10^?4m final concentration, added to suspensions of rabbit platelets prevented both the ADP-induced shape change and the increase in ³²P incorporation into PA and DPI. PGE₁, 10^?4m , also caused a decrease in labelling of TPI. PGE₁, 10^?7m final concentration, prevented platelet aggregation but did not prevent the ADP-induced shape change or the increase in ³²P-content of PA. Dibutyrylcyclic AMP (DBcAMP) inhibited both the ADP-induced shape change and the increase in ³²P-content of PA. With rabbit platelets, the time course of the ADP-induced shape change was compared to the time course of the increase in ³²P-content of PA, MPI and DPI. The increase in labelling of PA was detected 2 s after the addition of ADP and was near maximum at 8 s, the time of maximum shape change. The increase in labelling of DPI was not significant until 30 s. The regaining of the disc shape was associated with an increase in labelling of MPI and a decrease in labelling of PA and DPI. The results show that the changes in platelet PA are related to the ADP-induced shape change rather than to aggregation.     

Effect of active site-modified thrombin on the hydrolysis of platelet- associated glycoprotein V by native thrombin

To determine the relationship between equilibrium binding of thrombin to sites on the platelet surface and the cleavage of membrane glycoprotein V (GPV) by thrombin, we examined the effect of active site- modified thrombin (1-chloro-3-tosylamido-7-amino-L-2-heptanone thrombin toslysCH2-thrombin) on the binding of native thrombin to platelets and on the hydrolysis of GPV by native thrombin. ToslysCH2-thrombin inhibited binding of native thrombin to high affinity sites on the platelet surface. In contrast, hydrolysis of GPV by native thrombin, even at threshold thrombin concentrations, was not inhibited by pretreatment with toslysCH2-thrombin at concentrations up to 210 nmol/L. ToslysCH2-thrombin also had no appreciable effect on platelet aggregation or release of 14C-serotonin induced by native thrombin. Because toslysCH2-thrombin does not inhibit platelet release, aggregation, or GPV hydrolysis by native thrombin but does inhibit high affinity surface binding by native thrombin, these results indicate that thrombin binding and hydrolysis of GPV are separate and unrelated events.     

The metalloprotease,NN-PF3 from <Emphasis Type="Italic">Naja naja</Emphasis> venom inhibits platelet aggregation primarily by affecting α2β1 integrin

NN-PF3 is a non-toxic, anticoagulant, high-molecular-mass (67.81 kDa) metalloprotease from Indian cobra (Naja naja) venom. In the present study, NN-PF3 was investigated for the mechanism of inhibition of collagen-induced aggregation of human platelets. The complete inhibition of collagen-induced aggregation and partial inhibition of ADP- and epinephrine-induced aggregation has the respective IC₅₀ of 75 ± 5, 185 ± 10, and 232 ± 12 nM, whereas no inhibition of thrombin-, arachidonic acid-, and ristocetin-induced aggregation of platelets was observed in platelet-rich plasma. Further, native NN-PF3 and EDTA-inactivated NN-PF3 inhibited collagen-induced aggregation of washed platelets with respective IC₅₀ of 75 ± 4 and 180 ± 6 nM. The higher inhibitory effect of native NN-PF3 compared with EDTA-inactivated NN-PF3 suggests the enzymatic and non-enzymatic mechanism of inhibition. NN-PF3 pretreatment affected the collagen binding but not the fibrinogen, and fibronectin binding of washed platelets in adhesion assay suggested that the collagen receptors are affected. Western blot study using anti-integrin α2β1 mAb 6F1 suggested that NN-PF3 binds to integrin α2β1 in a primary structure-dependent manner only and is not cleaved. There was a drastic reduction in the intensity of several intracellular signaling phosphotyrosine protein bands when monoclonal anti-phosphotyrosine antibody was used, suggesting that the major activation pathway of platelets get affected, which occurs through glycoprotein VI. NN-PF3 did not bind to collagen as revealed by Western blot using anti-collagen mAb. Furthermore, neither the proteolytic cleavage of fibrinogen nor its degradation products by NN-PF3 contributed for the collagen-induced platelet aggregation inhibition.     

Preclinical Profile of PF9404C,a Nitric Oxide Donor with β Receptor Blocking Properties

PF9404C ((2'S),(2S)‐3‐isopropylamine, 1‐[4‐(2,3‐dinitroxy)propoxymethyl]‐phenoxy‐2′‐propranol) is the S‐S diesteroisomer of a novel blocker of β‐adrenergic receptors with vasorelaxing properties. It causes a concentration‐dependent relaxation of rat aorta helical strips precontracted with 10^?6 M norepinephrine (NE; IC₅₀ 33 nM). It is equipotent to nitroglycerin (NTG; IC₅₀ 49 nM), but much more potent than isosorbide dinitrate (ISD; IC₅₀ 15,000 nM). In rat aorta smooth muscle cells, at 10 μM, PF9404C increased the formation of cGMP from 3 pmol/mg protein in basal conditions to 53 pmol/mg protein, suggesting that the mechanism of its vasorelaxing effects involves the slow generation of NO. This is supported by the facts that (i) ODQ (a blocker of guanylate cyclase) inhibited the vasodilatory effects of PF9404C; and (ii) PF9404C generates NO, as indirectly measured by the Griess reaction. In the electrically driven guinea pig left atrium, PF9404C blocks the inotropic effects of isoproterenol in a concentration‐dependent manner. Its IC₅₀ (30 nM) was similar to that of S‐propranolol (22.4 nM) and lower than that of metoprolol (120 nM) or atenolol (192 nM). The β adrenergic ligand (‐)‐[³H]‐CGP12177 (4‐[3‐[(1,1‐dimethylethyl)amino]‐2‐hydroxypropoxy]‐1,3‐dihydro‐2H‐benzimidazol‐2‐one hydrochloride) (0.2 nM) is displaced from its binding sites in rat brain membranes with a K_i of 7, 17, 170, and 1200 nM for PF9404C, S‐(‐)propranolol, metoprolol, and atenolol, respectively. PF9404C blocks ⁴⁵Ca²⁺ entry into bovine adrenal chromaffin cells induced by direct depolarization with 70 mM K⁺ or by the nicotinic agonist dimethylphenylpiperazinium (DMPP). PF9404C exhibits about 3‐fold higher potency than NTG to relax the majority of the vessels studied, especially when they were contracted with K⁺, and shows a certain selectivity of action for the renal artery. It produces auto‐tolerance that is ca. 20‐fold less pronounced than that observed with NTG. Cross‐tolerance in preparations pre‐exposed to PF9404C and later relaxed with NTG, was much greater than auto‐tolerance. This makes PF9404C a useful pharmacological tool for the development of novel NO‐donor compounds with a lesser degree of vascular tolerance than those currently available.     

Characterization of the thromboxane synthase pathway product 12-oxoheptadeca-5(Z)-8(E)-10(E)-trienoic acid as a thromboxane A2 receptor antagonist with minimal intrinsic activity

Thromboxane synthase forms thromboxane (TX) A₂ and 12(S)-hydroxyheptadeca-5(Z)-8(E)-10(E)-trienoic acid (HHT) at equimolar amounts. Twelve-oxoheptadeca-5(Z)-8(E)-10(E)-trienoic acid (Oxo-HT) is the primary metabolite of HHT and has been described to be an inhibitor of platelet aggregation. Functional studies, Schild analysis and competitive binding studies were performed to clarify its mode of action. Oxo-HT was prepared biosynthetically as well as chemosynthetically, purified and characterized by gas chromatography and mass spectrometry. Platelet activation was assessed by determination of shape change, aggregation, fibrinogen binding and P-selectin expression using optical aggregometry and flow cytometry. Oxo-HT 0.1 n M to 50 μM did not induce platelet activation. Furthermore, it had no effect on platelet activation induced by thrombin, ADP or PAF. In contrast, Oxo-HT inhibited platelet aggregation, fibrinogen binding and P-selectin expression induced by U46619 in a competitive manner. Schild analysis for U46619-induced fibrinogen binding and P-selectin expression revealed pA₂ values of 6.1 and 6.6, respectively, which correspond to K_d values of approximately 0.8 μM and 0.3 μM , respectively. Oxo-HT also inhibited U46619 induced shape change (IC₅₀ ? 10 μM ). However, Oxo-HT over a concentration range of 0.1–1 μM enhanced the partial shape change induced by low concentrations of U46619. Thus Oxo-HT seems to possess a minimal agonistic potential, which alone is not sufficient to trigger a platelet activation but can enhance low levels of platelet activation. Oxo-HT blocked the binding of [³H]SQ 29548 in a concentration-dependent manner, whereas HHT did not displace [³H]SQ 29548. The K_d of Oxo-HT determined from competition binding studies was 7.7 μM , about 10–25-fold higher than the apparent K_d determined by Schild analysis. This discrepancy might be due to a desensitization of the TXA₂ receptor triggered by the minimal intrinsic activity of Oxo-HT. We conclude that Oxo-HT is a naturally occurring specific TXA₂ receptor antagonist with minimal intrinsic activity. Oxo-HT may contribute to the regulation of TXA₂-induced platelet activation in vivo.     

Platelet Antithrombins: Role of Thrombin Binding and the Release of Platelet Fibrinogen

The nature of platelet antithrombin was elucidated by comparison of thrombin binding and antithrombin activities of intact platelets and by purification of antithrombin from platelet lysates using glycerol osmotic lysis, ethanol precipitation and Sephadex gel filtration techniques. The major portion of the antithrombin and thrombin binding activity of intact platelets is lost after brief sonication. The antithrombin activity in destroyed platelets is found to be due to platelet fibrinogen. Treatment of platelets with PGE₁ (100 μg/ml) markedly inhibits (>80%) the release of platelet fibrinogen induced by thrombin. However, the PGE₁ treatment produced slight (<30%) but significant decrease of antithrombin activity of intact platelets, whereas the binding of thrombin to platelets was not affected by PGE₁ treatment. The amounts of thrombin bound to and inactivated by PGE₁-treated platelets at the same cell concentration are identical. The above results suggest that platelets contain at least two antithrombin activities. One, which accounts for the major portion of platelet antithrombin is mediated by thrombin binding to platelets. The other, which attributes to a lesser extent to platelet antithrombin activity, is due to the release of platelet fibrinogen. Also, antithrombin is readily demonstrated in a plasma medium indicating physiological significance of platelet antithrombin.     

Lysophosphatidic acid induces protein tyrosine phosphorylation in the absence of phospholipase C activation in human platelets

Lysophosphatidic acid is a biologically active phospholipid able to induce cell proliferation and platelet aggregation. In this study we investigated the biochemical mechanisms of platelet activation by lysophosphatidic acid. We found that lysophosphatidic acid stimulated the binding of the photoreactive GTP-analog 4-azidoanilido-[α³²P]GTP to a 40-kDa protein on platelet membranes. Moreover, lysophosphatidic acid induced the rapid decrease of the intracellular concentration of cAMP in intact platelets, indicating that this lipid activates platelets by binding to a membrane receptor coupled to the inhibitory GTP-binding protein Gi. In agreement with a receptor-mediated action, we found that platelet activation by lysophosphatidic acid underwent homologous desensitization. In the absence of extracellular CaCl₂, lysophosphatidic acid did not induce platelet aggregation, and did not stimulate phospholipase C. However, under the same conditions, lysophosphatidic acid produced the rapid tyrosine phosphorylation of several platelet proteins. This effect was not mediated by the formation of thromboxane A₂. Our results demonstrate that, in lysophosphatidic acid-stimulated platelets, activation of protein-tyrosine kinases occurs in the absence of phospholipase C activation and platelet aggregation, and may be directly related to the activation of the G-protein-coupled lysophosphatidic acid-receptor.     

Factor H binds to platelet integrin αIIbβ3

Factor H is a plasma protein that regulates activity of the alternative complement pathway in plasma and on cell surface. Binding of factor H to a cell surface protects that cell against complement-induced damage. Factor H binds to glycosoaminoglycans, surface-immobilized C3b, L selectin, and integrins such as α_Mβ₁ (a direct binding) or α_Vβ₃ (an indirect binding mediated through intermediary plasma proteins). We studied the binding of factor H to platelets and to integrin α_IIbβ₃ (glycoprotein IIb-IIIa), the most abundant integrin on platelets. We measured binding of purified factor H to platelets or heterologous cells expressing recombinant α_IIbβ₃ using flow cytometry. We also measured binding of factor H to α_IIbβ₃ in cell free systems using either surface plasmon resonance or enzyme-linked immunosorbent assay. We found that factor H directly binds to α_IIbβ₃ and this binding has a dissociation constant (Kd) of 131 ± 60.9 nM and is not dependent on active conformation of α_IIbβ₃ or on the presence of cations. Considering the high affinity of this interaction, the abundance of α_IIbβ₃ integrin on platelets, and the high concentration of factor H in plasma, α_IIbβ₃ provides a constitutive presence of factor H on platelets. Activation of platelets increases platelet-bound factor; however, this increase in binding of factor H cannot be explained by additional binding of factor H to α_IIbβ₃ and perhaps involves other binding sites for factor H on platelets.