Altered membrane fluidity and signal transduction in the platelets from patients of thrombotic stroke

Several earlier studies have implicated platelet activation with the pathogenesis of thrombotic stroke. In this report we have studied the changes in membrane physical microenvironment and signal transduction in the platelets obtained from the patients with thrombotic stroke. Aggregation induced by the synthetic agonist thrombin receptor-activating peptide was significantly enhanced (p < 0.001) in the platelets obtained from the patients. Steady-state fluorescence anisotropy measurements using diphenylhexatriene reflected a significant increase in membrane microviscosity from 3.315 (± 0.103) in the control to 4.600 (± 0.119) in the stroke. Proteins of relative mobilities of 131, 100, 47 and 38 kDa were found to remain phosphorylated on tyrosine in the resting platelets obtained from thrombotic stroke patients while they were not phosphorylated in the control counterparts. Besides, calpain, a calcium dependent thiol protease present in the platelets, was found to remain active in this disease as reflected from the proteolysis of calpain substrates. Taken together, these data indicated abnormal circulating platelets in the patients of thrombotic stroke, which could contribute to the etiopathogenesis of this disease.     

New diagnostic possibilities for the detection of thrombophilic states

A series of coagulation tests is described by which an increased thrombotic tendency is likely to be detected. These tests were carried out in 268 patients with venous thromboembolic disease and in 583 patients with arterial thrombotic manifestations. In venous thromboembolism alterations which point to hypercoagulability were found in 50% of all cases. Most frequent findings were a diminished availability of t-PA followed by low levels of Protein C, AT III and of Heparin cofactor II. In arterial thrombotic disease alterations of the clotting system were found in 77% of the patients. There was a high frequency of increased spontaneous platelet aggregation, of a diminished t-PA availability and of a combination of both alterations. Other abnormal results were rare with the exception of a diminished Heparin cofactor II which was found in 3% of the patients. The consequences which arise for prophylaxis and therapy when the defect is known are discussed.     

Paroxysmal nocturnal hemoglobinuria. Enhanced stimulation of platelets by the terminal complement components is related to the lack of C8bp in the membrane

Recently, a protein isolated from the membrane of human E, the so-called C8 binding protein (C8bp), has been described. C8bp is characterized as a 65-kDa protein that binds to C8 and inhibits the C5b-9-mediated lysis in a homologous system. In the present study, membranes of peripheral blood cells were tested for the presence of C8bp by SDS-PAGE and immunoblotting. In all cells a protein band reacting with anti-C8bp was seen, the Mr, however, was only about 50 kDa. To further analyze the 50-kDa protein, we isolated the protein by phenol-water extraction and isoelectric focusing from papain-treated platelets. The isolated protein behaved similar to the E-derived C8bp: it inhibited the lysis of model target cells by C5b-9. To examine the function of C8bp in platelets, we tested platelets from patients suffering from paroxysmal nocturnal hemoglobinuria (PNH). These platelets were deficient in C8bp, being in accordance with their higher lytic susceptibility in vitro. In response to sublytic C5b-9 doses, the PNH platelets released considerably more serotonin and thromboxane B2 than normal platelets. By addition of purified C8bp, the thromboxane B2 release was suppressed, indicating that C8bp not only restricts the lytic complement attack, but also regulates the C5b-9-mediated stimulation of target cells. Thus, lack of C8bp might not only result in enhanced hemolysis, but also in enhanced stimulation of platelets, which in turn might contribute to the thrombotic complications seen in some PNH-type III patients.     

Effects of various doses of SP 54 on fibrinolytic activity in patients with thrombotic diseases

SP 54 was synthetized more than 30 years ago, but the proper oral dosage has not been established yet. Therefore we gave two doses. 150 and 500 mg to patients suffering from various arterial and venous thrombotic diseases, for investigating the dose-response effect. 16 patients received 150 mg, 14 patients 500 mg and 10 in each group both doses. We could observe significant changes in ELT, WBL and plasminogen level, but the increase of fibrinolytic activity was not dose-dependent in most cases. It indicates that an individual dosage is needed. Therefore we suggest an "SP 54 loading test" before prescribing the drug.     

COVID-19 induces a hyperactive phenotype in circulating platelets

Coronavirus Disease 2019 (COVID-19), caused by the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has affected over 30 million globally to date. Although high rates of venous thromboembolism and evidence of COVID-19-induced endothelial dysfunction have been reported, the precise aetiology of the increased thrombotic risk associated with COVID-19 infection remains to be fully elucidated. Therefore, we assessed clinical platelet parameters and circulating platelet activity in patients with severe and nonsevere COVID-19. An assessment of clinical blood parameters in patients with severe COVID-19 disease (requiring intensive care), patients with nonsevere disease (not requiring intensive care), general medical in-patients without COVID-19, and healthy donors was undertaken. Platelet function and activity were also assessed by secretion and specific marker analysis. We demonstrated that routine clinical blood parameters including increased mean platelet volume (MPV) and decreased platelet:neutrophil ratio are associated with disease severity in COVID-19 upon hospitalisation and intensive care unit (ICU) admission. Strikingly, agonist-induced ADP release was 30- to 90-fold higher in COVID-19 patients compared with hospitalised controls and circulating levels of platelet factor 4 (PF4), soluble P-selectin (sP-selectin), and thrombopoietin (TPO) were also significantly elevated in COVID-19. This study shows that distinct differences exist in routine full blood count and other clinical laboratory parameters between patients with severe and nonsevere COVID-19. Moreover, we have determined all COVID-19 patients possess hyperactive circulating platelets. These data suggest abnormal platelet reactivity may contribute to hypercoagulability in COVID-19 and confirms the role that platelets/clotting has in determining the severity of the disease and the complexity of the recovery path.

The reason for the increased thrombotic risk associated with SARS-CoV-2 infection remains unclear. This study reveals that disease severity is associated with increased mean platelet volume and decreased platelet:neutrophil ratio; moreover, all COVID-19 patients possess hyperactive circulating platelets, with agonist-induced ADP release 30-to-90 fold higher than controls.     

Minor Role of Plasminogen in Complement Activation on Cell Surfaces

Atypical hemolytic uremic syndrome (aHUS) is a rare, but severe thrombotic microangiopathy. In roughly two thirds of the patients, mutations in complement genes lead to uncontrolled activation of the complement system against self cells. Recently, aHUS patients were described with deficiency of the fibrinolytic protein plasminogen. This zymogen and its protease form plasmin have both been shown to interact with complement proteins in the fluid phase. In this work we studied the potential of plasminogen to restrict complement propagation. In hemolytic assays, plasminogen inhibited complement activation, but only when it had been exogenously activated to plasmin and when it was used at disproportionately high concentrations compared to serum. Addition of only the zymogen plasminogen into serum did not hinder complement-mediated lysis of erythrocytes. Plasminogen could not restrict deposition of complement activation products on endothelial cells either, as was shown with flow cytometry. With platelets, a very weak inhibitory effect on deposition of C3 fragments was observed, but it was considered too weak to be significant for disease pathogenesis. Thus it was concluded that plasminogen is not an important regulator of complement on self cells. Instead, addition of plasminogen was shown to clearly hinder platelet aggregation in serum. This was attributed to plasmin causing disintegration of formed platelet aggregates. We propose that reduced proteolytic activity of plasmin on structures of growing thrombi, rather than on complement activation fragments, explains the association of plasminogen deficiency with aHUS. This adds to the emerging view that factors unrelated to the complement system can also be central to aHUS pathogenesis and suggests that future research on the mechanism of the disease should expand beyond complement dysregulation.     

Effect of streptokinase on the content of plasminogen activator inhibitor of type 1

The influence of streptokinase on plasminogen activators inhibitor of type I (PAI-1) was investigated with the use of model systems in vitro and in vivo. It was defined, that intravenous streptokinase injection causes an increase in PAI-1 content in mammals' blood plasma. Experiments in vitro have shown that the increase in PAI-1 concentration takes place as a result of streptokinase action. It occurs due to platelets activation with subsequent PAI-1 secretion from their a-granules. It is established, that PAI-1 is secreted by platelets both in free and in complex forms. The data obtained in the work, allow to assume, that the simultaneous substantial increase in PAI-1 content with platelets activation, as a result of streptokinase influence, can lead to new thrombotic complications risk.     

Hemostasis imbalance in experimental hypertension

BACKGROUND: The rat model of chronic intoxication by N(G) -nitro-L-arginine methyl ester (L-NAME) induces severe systemic arterial hypertension and progressive ischemic lesions in the central nervous system and kidneys. We investigated the possible molecular basis of these thrombotic events. METHODS AND RESULTS: Administration of L-NAME increased plasma markers of thrombin generation, thrombin-antithrombin complexes, and soluble glycoprotein V, measured by specific ELISA. Thrombin generation in vivo was associated with ex vivo platelet desensitization to adenosine 5'-diphosphate and collagen-induced aggregation. In the aortic layers and renal arterioles, tissue factor mRNA (semi-quantitative RT-PCR) and activity (coagulation assay) were increased. In contrast, tissue factor activity was not modified in glomeruli. In parallel, an impairment of the fibrinolytic system was demonstrated by an increase in plasma levels and arterial secretion of plasminogen activator inhibitor-1. In the arterial wall, plasminogen activator inhibtor-1 mRNA was significantly increased. Moreover, antifibrinolytic activity, studied by fibrin reverse zymography, was increased whereas all tissue-plasminogen activator activity secreted by the hypertensive arterial wall was detected as complexes with its specific inhibitor. In animals treated with the angiotensin-converting enzyme (ACE) inhibitor Zofenil, all of these parameters remained at control levels. CONCLUSIONS: These results indicate that chronic blockade of nitric oxide production in rats results in enhancement of blood markers of thrombin generation associated with tissue factor induction and impairment of fibrinolysis in the vascular wall, which may contribute to the thrombotic complications associated with hypertension.     

Decreased fibrinolytic activity in porcine-to-primate cardiac xenotransplantation.

BACKGROUND: One major barrier to successful xenotransplantation is acute vascular rejection, a process pathologically characterized by microvascular thrombosis and diffuse fibrin deposition in transplant blood vessels. This pathologic picture may result from a disturbance in the coagulant or fibrinolytic pathways that regulate normal vascular patency. This study evaluated the regulation of fibrinolytic activity defined by tissue plasminogen activator and plasminogen activator inhibitor-1 as it may exist in the setting of acute vascular rejection. MATERIALS AND METHODS, RESULTS: Serial biopsies from cardiac xenotransplants evaluated by immunofluorescence microscopy demonstrated progressive decreases in tissue plasminogen activator and increases in plasminogen activator inhibitor-1. In vitro studies measuring fibrinolytic activity of cell culture medium from porcine aortic endothelial cells stimulated with human serum or autologous porcine serum revealed that human serum triggered as much as 93% increase in antifibrinolytic activity. CONCLUSIONS: These findings demonstrate that porcine vascular endothelial cells change toward an antifibrinolytic state following stimulation with human xenoreactive antibodies and complement. The shift is at least partly explained by an increased ratio of plasminogen activator inhibitor-1 to tissue plasminogen activator, and is at least in part mediated by the activation of complement. This increased antifibrinolytic activity may contribute to the thrombotic diathesis seen in acute vascular rejection in pig-to-primate xenografts.     

Circulating platelet activation in healthy subjects and in some pathological conditions. Method of study and results

Current clinical methods for evaluating platelet function are artful tests which study the effects of various stimuli on platelets, whereas the clinician is much more interested in methods evaluating the activation of circulating platelets. The hallmark of activation of platelets is their shape change, i.e. the transformation of the platelets from smooth disks into spiny spheres; the aggregation begins when 30% of platelets are activated. In 1138 subjects (384 healthy individuals and 854 patients with various pathological conditions with high thrombotic risk) we have investigated circulating platelet activation and circulating platelet aggregates by fixation of blood cells in a glutaraldehyde mixture and by evaluation of platelet shape change and aggregates on a phase-contrast microscope. The method is precise, accurate and suitable for clinical purposes.     

Flow Cytometric Analysis of Circadian Changes in Platelet Activation Using Anti-Gmp-140 Monoclonal Antibody

The hemostatic activity of blood shows a circadian variation with a higher frequency of acute coronary events in the morning. The thrombotic tendency of blood is influenced by many factors, including platelets. Diurnal changes of in vivo platelet activation were investigated by whole blood flow cytometry in 10 young healthy male volunteers using anti-GMP-140 (anti-α-granule membrane protein 140 kD) monoclonal antibody at 3h intervals from 06:00 to 24:00. We also studied circulating platelet aggregates to investigate whether there exists a similarity between the results of these methods. Results of flow cytometric analysis indicate that there is an increase in platelet activation during the period from 06:00 to 09:00. Platelet activation then decreases gradually during the period from noon to midnight. These changes are accompanied by a similar trend in circulating platelet aggregates. This suggests that GMP-140 expression on platelets is synchronized with or followed by platelet aggregate formation in vivo, and increased platelet activation may predispose individuals to thrombosis at this time.     

Flow cytometric analysis of circadian changes in platelet activation using anti-GMP-140 monoclonal antibody.

The hemostatic activity of blood shows a circadian variation with a higher frequency of acute coronary events in the morning. The thrombotic tendency of blood is influenced by many factors, including platelets. Diurnal changes of in vivo platelet activation were investigated by whole blood flow cytometry in 10 young healthy male volunteers using anti-GMP-140 (anti-alpha-granule membrane protein 140 kD) monoclonal antibody at 3h intervals from 06:00 to 24:00. We also studied circulating platelet aggregates to investigate whether there exists a similarity between the results of these methods. Results of flow cytometric analysis indicate that there is an increase in platelet activation during the period from 06:00 to 09:00. Platelet activation then decreases gradually during the period from noon to midnight. These changes are accompanied by a similar trend in circulating platelet aggregates. This suggests that GMP-140 expression on platelets is synchronized with or followed by platelet aggregate formation in vivo, and increased platelet activation may predispose individuals to thrombosis at this time.     

Gray platelet syndrome: selective alpha-granule deficiency and thrombocytopenia due to increased platelet turnover

Clinical and laboratory studies of two siblings, both suffering from gray platelet syndrome (GPS) are described. The patients had a mild bleeding disorder, their platelets were blue-gray in panoptic stains, and alpha-granules were markedly reduced, as shown by electron microscopy. The platelet content of platelet factor 4 and that of beta-thromboglobulin were significantly reduced (3%-7% of normal). Platelet count was decreased (33-150 X 10(9)/1) and small platelets were increased in platelet volume distribution. Bleeding time was prolonged on most occasions. Bone marrow aspiration was performed in one patient and revealed increased reticulin fibers, however, megakaryocyte count was normal. The mean platelet survival was 4.8 days using 111indium-labelled platelets. In this patient, platelet-associated IgG was within the normal range. Prednisone therapy failed to increase platelet count. Dental surgery was performed under cover of desmopressin and no bleeding complication occurred; however, no improvement of bleeding time was observed. The patient delivered a healthy male infant without hemorrhaging while under concurrent platelet transfusion therapy.     

The role of insulin as an antithrombotic humoral factor

Insulin is well known for its essential role in carbohydrate metabolism: insulin deficiency results in the development of diabetes mellitus. It has been known for many years that people with diabetes mellitus are predisposed to develop thrombotic diseases including myocardial infarction. It was thought that the thrombus formation was the consequence of impaired carbohydrate metabolism. In recent years, it has become apparent that insulin is capable of ameliorating several pathophysiological events, leading to the inhibition and dissolution of the formed thrombus in the system. These insulin-induced events include inhibition of platelet aggregation by prompting the synthesis of NO in platelet and prostacyclin in endothelial cells. Furthermore, insulin upregulates prostacyclin receptors and downregulates alpha(2) adrenergic receptor in platelets, thereby amplifying the inhibition of platelet aggregation. Insulin also releases tissue plasminogen activator, a potent thrombolytic enzyme, from the platelet membrane which dissolves the formed thrombus leading to the resumption of normal blood circulation. In effect, insulin could be an essential tool in the control of thrombotic disorders.     

Antithrombotic therapy in patients with known risk factors for thromboembolism

In about 50% of the cases of spontaneous deep vein thrombosis a congenital deficiency of an inhibitor of coagulation or an insufficient fibrinolytic mechanism can be detected. In arterial thromboembolism a connection with hyperactive platelets or with a diminished availability of tissue plasminogen activator can be found in about 70%. However, in these cases the defect which provokes thrombosis is mostly acquired and is connected with hyperlipidemia and/or with atherosclerotic alterations of the vessel wall. A study on patients with thromboembolic tendency and detectable risk factors was carried out. A total of 470 patients could be observed for 2 years under an adequate antithrombotic prophylaxis. The occurrence of thromboembolic episodes 2 years prior to prophylaxis and 2 years under prophylaxis was compared. In venous cases thrombosis could be controlled almost completely by coumarins when the underlying cause was a deficient plasmatic inhibitor. In patients with diminished fibrinolysis there was only a partial effect of oral anticoagulants. A better result could be obtained when pentosan polysulfate was administered. In arterial thromboembolism the results of prophylaxis were less convincing. The efficacy of ASA in patients with an increased platelet function was only moderate. In addition, ASA hat to be discontinued in about 20% of the patients because of gastrointestinal problems. Pentosan polysulfate in patients with a diminished fibrinolytic capacity had a fairly good effect and resulted in a 60% reduction of thromboembolic manifestations. It is shown that an exact diagnosis of the underlying deficiency which is likely to cause thrombosis can also improve the efficacy and the specificity of prophylaxis.     

The effect of some epsilon-aminocaproic acid derivatives on platelet responses

epsilon-Aminocaproic acid (EACA) is a synthetic low molecular drug with antifibrinolytic activity. However, treatment with this drug can be incidentally associated with an increased thrombotic tendency. The aim of the present work was to test synthetic EACA derivatives for their antiplatelet activities. We investigated the effect of three EACA derivatives with antifibrinolytic activity: I. epsilon-aminocaproyl-L-leucine hydrochloride (HClH-EACA-L-Leu-OH), II. epsilon-aminocaproyl-L-(S-benzyl)-cysteine hydrochloride (HClH-EACA-L-Cys(S-Bzl)-OH) and III. epsilon-aminocaproyl-L-norleucine (H-EACA-L-Nle-OH) on platelet responses (aggregation and adhesion) and on their integrity. It was found that: 1. as judged by LDH release test, none of the tested compounds, up to 20 mM, was toxic to platelets, 2. in comparison with EACA, all the synthetic derivatives inhibited much stronger the ADP- and collagen-induced aggregation of platelets suspended in plasma (platelet rich plasma) and aggregation of these cells in whole blood, 3. EACA and its derivatives exerted a similar inhibitory effect on the thrombin-induced adhesion of platelets to fibrinogen-coated surfaces. Since platelet activation and blood coagulation are tightly associated processes, the antiplatelet properties of EACA derivatives are expected to indicate reduced thrombotic properties of these derivatives compared to EACA.     

Prevention of microvascular thrombosis with low-dose tissue plasminogen activator.

In a blind, randomized study, two groups, each of seven rabbits, were treated with either a very low dose of human melanoma cell line-derived tissue-type plasminogen activator (t-PA) or isotonic saline. t-PA (0.067 mg/kg of body weight) was administered intraaortically, 20 percent being given as a 30-second "bolus" infusion just prior to the reperfusion of intimectomized central ear arteries and the rest as a continuous infusion during the next 2 hours. Arteriotomic bleeding times, accumulations of 32P-labeled platelets, patency, and sizes of thrombus deposits 2 hours after reperfusion were recorded. To confirm the presence of tissue plasminogen activator in plasma, fibrin-plate lysis assays of arterial plasma were performed immediately before and 1/2 hour and 2 hours after starting drug infusion. Arteriotomic bleeding times were similar in both groups. Transient "oozing" from wound edges occurred in 40 percent of rabbits treated with tissue plasminogen activator. Patency was significantly increased and thrombus deposits were smaller in the tissue plasminogen activator group. Plasma from animals treated with tissue plasminogen activator caused massive lysis of fibrin plates, whereas plasma from control animals caused little or no lysis. Platelet accumulations were very similar in both groups, indicating that occlusive thrombi mainly consisted of other elements than platelets (e.g., fibrin and red cells). Scanning electron microscopy showed normally adhering and aggregating platelets in both groups. This study shows that mild fibrinolytic stimulation with tissue plasminogen activator significantly improves patency in severely traumatized small-caliber arteries and indicates that such treatment may be one approach to prevent thrombosis at microvascular anastomotic sites.     

Plasminogen activator activity in venous walls of patients who died of myocardial infarction

The aim of this study was to compare the activity of the plasminogen activator in walls of various veins obtained from 29 patients who died of myocardial infarction in the first 24 h. The control group consisted of 12 dissected persons without thrombotic complications before death. The activity of the plasminogen activator was determined by using the chromogenic substrate S-2251 according to SHIMADA technique. The twofold higher plasminogen activator activity was found in vessels of patients who died of myocardial infarction in comparison with persons who died of other causes. A significant correlation between the plasminogen activator activities of veins obtained from different parts of the body was also stated.     

Binding and activation of plasminogen on the platelet surface

A mechanism by which platelets might participate in fibrinolysis by binding plasminogen and influencing its activation has been examined. Binding of radioiodinated human Glu-plasminogen to washed human platelets was time-dependent and was enhanced 3-9-fold by stimulation of platelets with thrombin but not with ADP. The interaction with both stimulated and unstimulated cells was specific, saturable, divalent ion-independent, and reversible. The platelet-bound ligand had the molecular weight of plasminogen, and no conversion to plasmin was detected. Scatchard analyses provided evidence for a single class of plasminogen-binding sites on both stimulated and unstimulated cells. The Kd for thrombin-stimulated platelets was 2.6 +/- 1.3 microM, and 190,000 +/- 45,000 molecules were bound per cell, whereas unstimulated platelets bound 37,000 +/- 10,500 molecules/cell with a Kd of 1.9 +/- 0.15 microM. Plasminogen binding was inhibited in a dose-dependent manner by omega-aminocarboxylic acids at concentrations consistent with a requirement for an unoccupied high affinity lysine-binding site for plasminogen binding to the cells. When platelet-bound plasminogen was incubated with tissue plasminogen activator, urokinase, or streptokinase, gel analysis established that plasmin was preferentially associated with the platelet relative to the supernatant. Plasminogen and plasmin interacted with thrombin-stimulated platelets with similar binding characteristics, and there was no evidence for a binding site for plasmin which did not also bind plasminogen. Therefore, the results suggest that plasminogen activation is enhanced on the cell surface. In sum, these results indicate that platelets bind plasminogen at physiologic zymogen concentrations and this interaction may serve to localize and promote plasminogen activation.     

Role of platelets in hemostasis and thrombosis.

Platelets are central to both normal hemostasis and abnormal thrombotic states along with the vessel wall, coagulation elements, and blood flow. The platelets play a pivotal role in the reaction that occurs after vessel injury, during which platelets first adhere to the vessel wall, undergo a release reaction and then aggregate, probably as a result of the materials released from platelets. These processes can be studied by a series of in vitro tests which form the basis of our knowledge of platelets in hemostasis. While the hemostatic plug is usually microscopic in size, this same plug (platelet thrombus) may contribute to the pathogenesis of several arterial diseases such as transient ischemic attacks, sudden blindness, sudden cardiac death and acute respiratory death syndrome. Careful microscopic examinations have shown that platelet aggregates may be found in the microcirculation which could affect vital structures such as the conduction system of the heart. Both anatomic and therapeutic evidence evidence suggests that platelets play a role in venous thrombosis. Recent evidence suggests increased levels of materials known to be released from platelets in patients with both arterial and venous thrombi along with increased platelet coagulant activities in patients with venous thrombosis.