Inhibition of activated protein C by benzamidine derivatives

In studies on the inhibition of activated protein C (APC) by benzamidine derivatives potent inhibitors of APC were found among anilides of 4-amidinophenyl--aminobutyric acid (K_i = 0.58 μmol/1). Several bis-benzamidine derivatives containing a cycloalkanone linking bridge inhibit APC with K_i values near the micromolar range.

Potent and selective inhibitors of thrombin derived from 3- and 4-amidinophenylalanine do not inhibit APC. This is of great importance for further development of these inhibitors as potential anticoagulant drugs.     

Inhibition of cyclooxygenase-independent platelet aggregation by sodium salicylate

The effect of acetylsalicylic acid (ASA) on platelet aggregation (PA) and thromboxane A₂ (TxA₂) formation was investigated in vitro and ex vivo after 1 g or 300 mg ASA administration to healthy subjects. 50–100 μM ASA inhibited PA by single aggregating agent such as platelet aggregating factor (PAF) or epinephrine and reduced to 5% of control platelet TxB₂ formation, but did not influence PA by epinephrine plus PAF. The latter was inhibited by increasing ASA concentration. In samples incubated with 100 μM ASA and stimulated with epinephrine plus PAF, PA could be inhibited by the addition of 100–300 μM sodium salicylate. After 300 mg-1 g ASA administration to healthy subjects, the inhibition of PA by epinephrine plus PAF was more marked by highest doses of ASA. This study suggests that aspirin inhibits PA with a cy clooxygenase-independent mechanism; this effect is mediated, at least in vitro, by salicylic acid.     

Plasma albumin is essential for collagen-induced platelet aggregation

We found that platelets must have albumin on the surface to respond to collagen and aggregate. Albumin, however, was not absolutely necessary for ADP-, platelet activating factor-, serotonin- or thrombin-induced aggregation, while fibrinogen was required for ADP- or serotonin-induced aggregation. Immunofluorescent microscopy revealed that albumin was retained on gel-filtrated platelets but not on washed platelets. Albumin was not required for platelet adhesion to immobilized collagen. Without albumin thromboxane formation upon collagen-stimulation was diminished. These data suggest that albumin is essential in some step(s) that results in production of thromboxane A₂.     

Dual effect of naloxone on blood platelet aggregation and cerebral blood flow in gerbils

The effect of naloxone on blood platelet aggregation and cerebral blood flow in gerbils was studied. Administration of naloxone in dose 1 mg/kg to intact gerbils resulted in a marked increase in platelet aggregability accompanied by 27% reduction in cerebral blood flow. Focal cerebral ischemic injury significantly enhanced platelet aggregatory response and treatment with naloxone was without any additional effect on platelet aggregation. Cerebral blood flow in ischemic hemisphere, however, increased following naloxone injection by 46%. In vitro naloxone in millimolar concentrations inhibited platelet aggregation in a dose-dependent way. Apparent decrease in fluorescence of platelet membranes tagged with fluorescence probe due to naloxone suggests conformational changes in platelet membrane as a primary mechanism for the antiaggregatory effect of naloxone in vitro.     

Interactions between sodium salicylate and acetyl salicylic acid evaluated using ADP induced platelet aggregation and bleeding time

1 g acetyl salicylic acid orally significantly enhanced the initial rate of platelet aggregation induced by 1 pmol/1 and 2.5 μ mol/1 ADP.

Sodium salicylate was without effects on the platelet aggregation and specifically it did not prevent acetylsalicylic acid from inhibiting the secondary aggregation. Sodium salicylate was without effect on the bleeding time and did not inhibit the prolongation induced by acetyl salicylic acid. Our study do not lend support to the concept of an important interaction in vivo between acetyl salicylic acid and its first metabolite salicylate in man.     

Inhibitory effect of vitamin E (α -tocopherol) on spontaneous platelet aggregation in whole blood

Vitamin E (D-α-tocopherol) inhibited spontaneous human platelet aggregation in whole blood in the 20–200 μ g/ml range. When α -tocopherol (20 μ g/ml) and aspirin (0.5 mM), or α -tocopherol and the mixture of phosphocreatine (1.5 mM) and creatine phosphokinase (50 U/ml) (CP/CPK) were added to this reaction system, a synergic inhibitory effect on aggregation was observed. On the other hand, when both α -tocopherol and the specific inhibitor of platelet activating factor (CV-3988 : 0.38 mM) were added to this system, the inhibition was the same as that caused by the addition of CV-3988 alone, suggesting there was no synergism, i.e., that the effect of α -tocopherol is related to the inhibition of platelet activating factor (PAF)-induced platelet aggregation in whole blood. However, α -tocopherol (20 or 50 μ g/ml) did not inhibit PAF (10 nM) induced platelet aggregation in platelet rich plasma (PRP). These results suggest that the inhibition of platelet aggregation in whole blood by α -tocopherol is due to the inhibition of PAF synthesis, and is unrelated to adenosine diphosphate (ADP) or thromboxane A2.     

The primary wave of epinephrine-induced platelet aggregation represents alpha 2-adrenoceptor status

We examined relationships between epinephrine-induced slope of primary wave of aggregation and the alpha₂-adrenoceptor status on platelets. A concentration (10^-9 to 10^-6 )-dependent increase in slope of primary wave with EC50 of epinephrine at 4.5 ± 0.4 × 10^-7 was observed. In studies on epinephrine binding to alpha₂adrenoceptors in competition with ³H-yohimbine to platelets, (IC₅₀) of epinephrine was 4.8 ± 3.4 x 10^-7 M. There was a significant (P<0.02) correlation between EC₅₀ of epinephrine to evoke biological response and IC₅₀ of epinephrine to bind to alpha₂-adrenoceptors (r-0.75). There was no relationship between number of receptor sites or dissociation constant of ³H-yohimbine binding and primary wave of platelet aggregation. These data show that the slope of primary wave in response to epinephrine reflects alpha₂-adrenoceptor binding of the agonist.     

Electron microscopic study of platelet agglutination induced by thrombotic thrombocytopenic purpura plasma containing 37-KDa platelet agglutinating protein

It has been demonstrated that plasma from a patient with thrombotic thrombocytopenic purpura (TTP) and 37-KDa platelet-agglutinating protein (PAP p37) purified from the same plasma caused the agglutination of platelets from normal subjects as well as from the same patient after recovery without the requirement of extracellular Ca⁺⁺ and fibrinogen. Experiments were designed to study the morphologic changes of platelets as a result of agglutination and the distribution of platelet receptors for PAP p37 under transmission electron microscope. Following incubation with TTP plasma or PAP p37 with stirring, platelets showed shape change, pseudopod formation, variable degrees of degranulation, dilatation of open canalicular systems and formation of agglutinates composed of a few to several hundred platelets. After platelets were incubated with TTP plasma or PAP p37 they were washed and further incubated with rabbit anti-PAP p37 serum without stirring followed by immuno-staining. Abundant electron dense reaction products were bound directly and randomly to the outer surface of the membrane of solitary platelets. When the reaction mixture was stirred, electron dense particles were also present between the platelet membranes in the agglutinates. No staining was observed in control experiments using normal plasma or non-immune rabbit serum. These results indicate that the TTP plasma containing PAP p37 causes agglutination, shape change, and variable degrees degranulation in platelets and that PAP p37 binds randomly to the outer surface of platelet membrane.     

Species differences in platelet responses to thrombin and SFLLRN. receptor-mediated calcium mobilization and aggregation, and regulation by protein kinases

The thrombin receptor on human platelets is activated by thrombin to stimulate platelet aggregation through the tethered ligand SFLLRN. This study examined the effects of thrombin and SFLLRN on aggregation and calcium mobilization ([Ca²⁺]_i) in rat, guinea pig, rabbit, dog, monkey, and human platelets, and the role of protein kinases in regulating these functions. Thrombin induced platelet aggregation and [Ca²⁺]_i in all species studied; however, only guinea pig, monkey and human platelets were responsive to SFLLRN. Similar species specific effects were obtained with [Ca²⁺]_i studies. The kinetic profile for [Ca²⁺]_i differed among species, suggesting that regulatory mechanisms for calcium differed between agonists and among species. Staurosporine, a non-selective inhibitor of protein kinases, inhibited platelet aggregation induced by thrombin or SFLLRN in all species. Staurosporine inhibited thrombin-induced [Ca²⁺]_i in guinea pigs, had no effect in rat, and increased [Ca²⁺]_i in all other species. Staurosporine inhibited SFLLRN-induced [Ca²⁺]_i in guinea pig, yet had no effect in monkey or human. Tyrphostin 23, a specific inhibitor of tyrosine protein kinases, inhibited thrombin-induced aggregation of rabbit, monkey, dog and human platelets. SFLLRN-induced aggregation was also inhibited by tyrphostin 23. Tyrphostin 23 inhibited [Ca²⁺]_i induced by either thrombin or SFLLRN in all species. Based on the differential response to agonist stimulation, we propose that thrombin can activate platelets via SFLLRN-dependent and independent mechanisms, which could involve yet unrecognized subtypes of the thrombin receptor or distinct cellular activating mechanisms. Furthermore, differential regulation of calcium mobilization and aggregation was observed in those platelets responding to either thrombin or SFLLRN.     

The influence of sample age on collagen-induced platelet aggregation in whole blood

Whole blood electrical aggregometry (WBEA) has become an accepted method to gain quick information on platelet disorders. Compared to the optical method WBEA is closer to physiology and less complicated, but on the other hand more difficult to standardize. Different approaches have been attempted in the past to improve the reliability and practicability of this technique. The influence of sample age has not been defined so far. In a first step a mathematical modelling program was established, which is able to characterize the aggregation curves obtained after collagen stimulation. In a mathematical analysis various characteristics of the curve function were calculated and their sensitivity for aging investigated. Regression was performed for each characteristic, and correction factors defined.

Our results indicate, that whole blood specimen for collagen induced aggregation can be used without correction factor up to 30 minutes. Data obtained with an age exeeding half an hour have to be corrected following a quadratic regression.     

Effect of beta-pyridyl-carbinol on platelet aggregation

We studied the anti-platelet aggregation activity of beta-pyridyl-carbinol (b-PC) (Ronicol, Roche). This compound has a chemical structure similar to nicotinic acid and is therapeutically indicated in functional and organic circulatory processes.

We determined the in vitro antiaggregation activity induced by ADP (4 μM) and collagen (20 μg/ml) using Cardinal and Flower's technique.

Antithrombotic activity was assessed in rats by measuring the duration of ADP-induced (12.40 mg/kg i.v.) respiratory dysfunction in 2 groups: one given 17.14 mg/kg beta-pyridyl-carbinol for 8 consecutive days before testing and controls.

In vitro and in vivo results were highly significant. Circulating platelet count increased in b-PC treated animals.

Systolic and diastolic pressures remained unmodified during administration of b-PC.     

Action of some salicylate derivatives on in vitro platelet aggregation. Inhibitory and inhibition antagonistic effects

Twenty salicylate derivatives were tested for their antagonistic activity on the inhibitory effect of aspirin on platelet aggregation. The blocking effect was not limited to the salicylate but also characterised some of its substituted compounds. The substituant influence did not seem to be related to electronic or size parameters. This antagonistic activity of these derivatives decreased as concentrations increased, owing to the emergence of their own inhibitory activity: several salicylate derivatives showed dual inhibitory and inhibition antagonistic activity, with both properties present at the same concentration. A mechanism involving dissociated activities on the two enzymatic sites of cyclooxygenase is proposed.     

Involvement of calmodulin in platelet reaction

Penothiazines, known as selective inhibitors of calmodulin, completely inhibited platelet aggregation and secretion induced by ADP, collagen, epinephrine, thrombin or calcium ionophore. They also completely inhibited aggregation induced by exogenous arachidonate (AA) or a mixture of thromboxane A₂ and prostaglandin endoperoxides (TxA₂/PG G₂,H₂). Also, in the presence of these calmodulin inhibitors, the release of AA from platelet phospholipids (PL) was dosedependently inhibited in stimulated platelets. These observations suggest that in platelet reaction, calmodulin is involved in at least two different steps of the reaction: activation of phospholipases and contraction of platelet actomyosin after the formation of TxA₂.     

Glycoprotein V hydrolysis by thrombin. Lack of correlation with secretion

Platelet membrane glycoprotein V (GPV) was hydrolyzed during thrombin-induced platelet aggregation releasing a fragment GPV_f1 into the supernatant. Hydrolysis of GPV required catalytically active thrombin and was diminished by chemical modification of the fibrinogen binding site of thrombin. Half maximal liberation of GPV_f1 occurred at a 10-fold higher concentration of thrombin than was required for half-maximal release. Time course studies at several thrombin concentrations showed disparate release of GPV_f1 and thrombospondin. These results emphasize the complexity of the initial events in thrombin-induced platelet activation.     

The effect of myelin basic protein on the endogenous phosphorylation of platelets

Bovine myelin basic protein (BP) induced a shape change and endogenous phosphorylation of a 45,000 (45K) molecular weight protein of intact human platelets. This effect occurred rapidly over an effective concentration range of 5–100 uM BP. BP peptides encompassing residues 1–42, 43–88, and 89–169 from the BP molecule of 169 residues, neither induced phosphorylation of platelets nor blocked the effect of intact BP. Subfractionation of disrupted platelets demonstrated the phosphorylated 45K protein in the 100,000 xg supernate. When isolated platelet membranes were used. no BP induced phosphorylation of a 45K protein could be detected. The amino acid composition of the purified, phosphorylated 45K protein differed from those of other known platelet proteins. BP itself was also phosphorylated by an endogenous platelet protein kinase(s) present both in the 100,000 xg supernatant and in the isolated membrane fraction of platelets. These results indicate that the normal or pathological release of BP from myelin may lead to phosphorylation of an internal protein of platelets and possibly other tissue elements with resultant metabolic and functional changes.     

Determination of functional and antigenic protein C inhibitor and its complexes with activated protein C in plasma by ELISA''s

Enzyme-linked immunosorbent assays (ELISA's) were developed for the measurement of protein C inhibitor (PCI) antigen and activity and for its complexes with activated protein C (APC) in plasma. For PCI activity and antigen, APC or anti-PCI, respectively, was immobilized to microtiter plates and PCI bound was detected with labelled anti-PCI antibodíes. For APC:PCI complexes, two different antibodies directed against protein C and PCI were used. The assays for PCI were calibrated with pooled normal human plasma (NHP) and with purified PCI, and for APC:PCI complexes with known concentrations of purified pre-formed complexes added to buffer or to plasma. The lower limit of sensitivity of the PCI activity and antigen assays was 10 ng/ml and 0.5 ng/ml, respectively and for plasma APC:PCI complexes 12 ng/ml. Mean coefficients of variation of 1.5 % to 5.8 % (intro-assay) and 2.1 % to 9.8 % (inter-assay) were found for the assays. For PCI antigen, a range of 56 % to 162 % of the NHP value was obtained in samples from 70 healthy donors (mean ±SD = 98.6 % ±23.1%). For PCI activity, the range was 59 % to 148 % (94.3 % ± 20.2). A good correlation (0.92) was obtained when both assays were compared. Plasma levels of APC:PCI complexes in 30 normals were under the detection limit (< 12 ng/ml). In plasma samples from 10 patients with disseminated intravascular coagulation (DIC) PCI antigen concentrations were decreased (55.6% ±20%) and 8 of the patients had APC-PCI complex levels between 32 and 240 ng/ml (median, 35 ng/ml). After addition of 20 ωg/ml APC to NHP or to protein C depleted plasma, 6.1 μg/ml complexes were recovered after 90 min incubation. Incubation of 10 μg/ml APC with NHP in the presence of 10 U/ml heparin yielded 11 μg/ml complexes after 90 min, which represent more than 90 % of the maximum possible value. Thus, the method should be adequate to study complexes of APC in vivo in clinical conditions in which activation of protein C pathway may occur.