Dependence of haemostasis system response from initial blood coagulation activity under total joints replacement

Effect of the initial state of the plasma hemostasis on the hemocoagulation changes after the total arthroplasty surgery was studied in 100 patients with osteoarthritis. Indicators of coagulation, fibrinolysis, and physiological anticoagulants were determined before and after completion of the surgery, at days 1, 3, 7, and 13-14 postoperatively. Increased coagulation activity befor surgery enhanced blood clotting within three days after the surgery. Enhanced consumption of physiological anticoagulants reduced the ability to recover their level a week after arthroplasty. The raised activity of the fibrinolysis inhibitor retained the effect during three postoperative days. Initial abnormalities in plasma hemostasis enhance blood coagulation dysfunction caused by surgical intervention on the large joints.     

Hemostatic mechanisms and malignant tumors

The mutual relationships between malignant tumours and mechanisms of blood coagulation are presented in a brief survey. In this connection, the mechanisms of a tumour cell entering the circulation through the vessel well and its leaving into the tissues are discussed, the theory of microtrauma being used for explaining these processes. Subsequently, the alterations to be found in the count and function of thrombocytes after contact with a malignant cell and the impact on this cell by blood platelets are represented. As a third factor the activation of blood coagulation which is exercised by substances with a procoagulatory effect produced by the malignant tissue and the frequently observed thrombosis in the course of neoplastic diseases are dealt with in connection with blood level changes of some coagulation factors. In a fourth section the significance of fibrinolysis, its activation and inhibition as well as the production of fibrinolytic activators by neoplasms are discussed.     

Red cell 2,3-DPG, ATP, and mean cell volume in highly trained athletes. Effect of long-term submaximal exercise

20 male elite long distance runners were compared to a control group of blood donors to determine the effect of training on red blood cells. The acute effects of exercise on red cells were investigated in 11 of the runners following a race of 15-30 km. The runners had elevated resting values of red cell 2,3-DPG (P less than 0.05) and mean cell volume (P less than 0.01); blood Hb and ATP were not different from concentrations in the control group. The red cell status of the athletes may be explained by an increased proportion of young erythrocytes in runners. No statistically significant changes in red cell 2,3-DPG, ATP, mean cell volume or blood Hb were found post exercise.     

Increases in platelet and red cell counts,blood viscosity,and arterial pressure during mild surface cooling: factors in mortality from coronary and cerebral thrombosis in winter.

Six hours of mild surface cooling in moving air at 24 degrees C with little fall in core temperature (0.4 degree C) increased the packed cell volume by 7% and increased the platelet count and usually the mean platelet volume to produce a 15% increase in the fraction of plasma volume occupied by platelets. Little of these increases occurred in the first hour. Whole blood viscosity increased by 21%; plasma viscosity usually increased, and arterial pressure rose on average from 126/69 to 138/87 mm Hg. Plasma cholesterol concentration increased, in both high and low density lipoprotein fractions, but values of total lipoprotein and lipoprotein fractions were unchanged. The increases in platelets, red cells, and viscosity associated with normal thermoregulatory adjustments to mild surface cooling provide a probable explanation for rapid increases in coronary and cerebral thrombosis in cold weather. The raised arterial pressure and possibly cholesterol concentration may contribute to slower components of the increased thrombosis.     

Streptokinase and Deep Vein Thrombosis

Five patients with deep vein thrombosis were given streptokinase. Five others with similar phlebograms were given anticoagulants, and the results assessed by examining changes in the iliac, femoral, and calf segments of the phlebograms when repeated 7-10 days later. Those of the anticoagulant group were unchanged. Four of the five given streptokinase had a reduction in the size and extent of their thrombosis. Fresh thrombus surrounded by flowing blood was lysed. Thrombus completely blocking a vein was not lysed.The indications for the use of streptokinase in deep vein thrombosis are reviewed.     

Relationships between hemorheology, erythrocyte metabolism, and von willebrand factor in athletes and patients with peripheral arterial disease

The relationship between hemorheology, erythrocyte ATP and 2,3-diphosphoglycerate (2,3-DPG) concentrations, and von Willebrand factor antigen was studied in athletes and peripheral arterial disease patients. Lower blood viscosity, mainly due to a higher erythrocyte deformability, was found in athletes compared to control subjects. Higher 2,3-DPG/Ht levels in athletes were correlated with blood viscosity, erythrocyte deformability, the rigidity index, and erythrocyte suspension viscosity at low shear stress. It is suggested that these relationships might be determined by the predominance of immature erythrocytes in the blood circulation of the athletes. In the group of patients, a decrease in ATP/Ht was related to increased erythrocyte aggregation and a higher erythrocyte suspension viscosity. Moreover, the concentration of von Willebrand factor was positively correlated with the erythrocyte aggregation index, erythrocyte suspension viscosity, and plasma viscosity. The results show that alterations in erythrocyte and plasma rheology may be involved in the modification of the functional state of the vascular endothelium and the development of atherosclerosis.     

Effects of compounds from Passiflora edulis Sims f. flavicarpa juice on blood coagulation and on proteolytic enzymes

Passion fruit (Passiflora edulis Sims f. flavicarpa) is popularly known for its sedative and calming properties and is consumed as a fresh fruit or as a juice. The clinical observation of blood incoagulability associated with excessive consumption of passion fruit juice, in a patient treated with warfarin, prompted the current study to investigate in vitro the presence of blood clotting inhibitors in Passiflora edulis Sims f. flavicarpa extract. After purification process, two compounds of distinct molecular weight and inhibitory action were better characterized. One is a trypsin inhibitor similar to inhibitors from Bowman-Birk family, named PeTI-I12, and other is a compound active in coagulation that prolongs aPTT and PT, but does not change TT. The aim of this study is to provide evidence that passion fruit extract's components play a role on hemostasis and therefore may be relevant in the handling of patients treated with anticoagulants or suffering hemorrhagic diseases.     

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.     

G13 is an essential mediator of platelet activation in hemostasis and thrombosis

Platelet activation at sites of vascular injury is essential for primary hemostasis, but also underlies arterial thrombosis leading to myocardial infarction or stroke. Platelet activators such as adenosine diphosphate, thrombin or thromboxane A(2) (TXA(2)) activate receptors that are coupled to heterotrimeric G proteins. Activation of platelets through these receptors involves signaling through G(q), G(i) and G(z) (refs. 4-6). However, the role and relative importance of G(12) and G(13), which are activated by various platelet stimuli, are unclear. Here we show that lack of Galpha(13), but not Galpha(12), severely reduced the potency of thrombin, TXA(2) and collagen to induce platelet shape changes and aggregation in vitro. These defects were accompanied by reduced activation of RhoA and inability to form stable platelet thrombi under high shear stress ex vivo. Galpha(13) deficiency in platelets resulted in a severe defect in primary hemostasis and complete protection against arterial thrombosis in vivo. We conclude that G(13)-mediated signaling processes are required for normal hemostasis and thrombosis and may serve as a new target for antiplatelet drugs.     

Liposome-Encapsulated Hemoglobin: A Red Blood Cell Substitute

Abstract

A safe and effective red blood cell (RBC) substitute would have broad implications in the practice of emergency medicine, trauma management, surgery, and several other areas of medicine. Several hemoglobin-based RBC substitutes have been developed that can deliver oxygen to peripheral tissues. However, although these RBC substitutes have desirable biophysical properties, their in vivo efficacy is limited by their significant toxicity. In view of the very high doses of blood substitute that are likely to be used clinically, toxicity as well as other safety issues that include hemostasis and thrombosis are critical considerations for the development and ultimate application of RBC surrogates.

Recent work conducted in our laboratories has demonstrated that administration of liposome-encapsulated hemoglobin (LEH) in rats was efficacious. Also our results have demonstrated that the replacement of more conventional lipids with the sterically-stabilizing lipid polyethyleneglycol distearoylphosphatidyl-ethanolamine in the LEH results in a significant decrease in LEH immunotoxicity, as measured by host resistance to infectious insult.     

Intradimer forces and their implication for conformations of von Willebrand factor multimers

The largest blood glycoprotein von Willebrand factor (VWF) responds to hydrodynamic stresses in the bloodstream with abrupt conformation changes, thus increasing its adhesivity to platelets and collagen. Arterial and microvascular hemostasis relies on mechanical and physicochemical properties of this macromolecule. Recently, it was discovered that the mechanical properties of VWF are controlled by multiple pH-dependent interactions with opposite trends within dimeric subunits. In this work, computer simulations reveal the effect of these intradimer forces on the conformation of VWF multimers in various hydrodynamic conditions. A coarse-grained computer model of VWF has been proposed and parameterized to give a good agreement with experimental data. The simulations suggest that strong attraction between VWF D4 domains increases the resistance to elongation under shear stress, whereas even intermediate attraction between VWF C domains contributes to VWF compaction in nonsheared fluid. It is hypothesized that the detailed subdimer dynamics of VWF concatamers may be one of the biophysical regulators of initial hemostasis and arterial thrombosis.     

The Role of the Initial State in the Response of the Hemostasis System to Double Exercise

Changes in the hemostasis system were studied in males with a high working capacity when they performed the same dosed double stress exercise test with moderate (N ₁ = 1.4 W/kg, t ₁ = 6.93 min) and high (N ₂ = 2.96 W/kg, t ₂ = 3.15 min) intensity with a 1-h interval between. The direction and the magnitude of changes in the main variables of blood coagulation, anticoagulants, and blood and plasma fibrinolytic activity under the influence of exercise were shown to correlate with the initial state of the system. At high values of the variables before the beginning of muscular activity, work results in their decrease, and at low values, in their increase. Previous work increases the dependence of changes in blood coagulation on its initial value upon repeat exercise, without influencing the relationship between alterations in the fibrinolytic activity of whole blood and its value before exercise. Double loading reveals closer relations between the initial values of blood coagulation, anticoagulants, and fibrinolysis and their changes due to work. It was concluded that, on the basis of the correlations presented, it is possible to predict with high probability the direction and the magnitude of hemocoagulant and fibrinolytic response to physical activity in the group of subjects studied.     

Endothelial dysfunction in rectal cancer patients chronically exposed to ionizing radiation

We sought to identify the features of endothelial function in rectal cancer patients who were exposed to chronic ionizing radiation from a nuclear test site in Kazakhstan. We examined 146 individuals, 76 of whom were rectal cancer patients. The existence of a complex of disturbances of the endothelium and hemostasis systems in patients vs non-patients was revealed. Endothelial dysfunction was expressed as an increase of nitric oxide (NO) production along with decreases in vasodilatation function, and increased levels of von Willebrand factor in blood, along with an increase in the number of circulating endotheliocytes. Significant correlations between indicators of endothelial function and vascular-platelet hemostasis were observed. These changes and their interrelations were expressed more strongly in the patients who lived in the contaminated area around the nuclear test site. Such patients could have an increased risk of thrombosis and other complications after the treatment of a malignant neoplasm.     

Antiphospholipid antibodies in young myocardial infarction patients

Myocardial infarction (MI) is a multi-factorial disease which claims many young lives. There are very few Indian studies that have investigated antiphospholipid antibodies (APLs) in MI patients. APLs have been implicated in arterial thrombosis including premature coronary artery and cerebrovascular thrombosis. In the present study, the prevalence of two clinically significant APLs--anticardiolipin antibody (ACA) and lupus anticoagulants (LA) in young MI patients was studied and compared with age- and sex-matched controls. Fifty healthy blood donors and 40 young MI patients (less than 45 yrs) diagnosed according to the American Heart Association guidelines were recruited for the study. The criteria for diagnosis were presence of atleast two of three classical findings including: clinical symptoms, diagnostic ECG, and presence of one or more cardiac biomarkers out of raised CK-MB isoform and T-troponin on serial measurement. LA and ACA were tested by lupus-sensitive activated partial thromboplastin time (aPTT) and ELISA respectively. Elevation of ACA was observed in 9 patients, while 6 were positive for LA. ACA of IgG isotype was detected in 8 patients. One patient had LA and raised ACA of IgG and IgM isotypes. Antiphospholipid antibodies were found to be significantly associated with MI in young patients, when considered together (p < 0.05) and in coronary thrombosis, mild elevation of ACA may be considered significant.     

Effect of strength training on pressor reflex responses from receptors in exercising muscles

The effect of strength training on muscle pressor reflex responses was investigated. Ten young, healthy volunteers and eight arm wrestling athletes performed forearm exercises at 30% of maximal voluntary effort until exhaustion. The exercises were either static or rhythmic, with alternating 20-s periods of muscle contraction and relaxation, followed by postexercise forearm arterial occlusion (PEAO). Heart rate, blood pressure (BP), and sympathetic nerve activity directed to muscle blood vessels (MSNA) were continuously recorded during the exercises. MSNA recordings were obtained from the peroneal nerve using a microneurographic method. During static exercises followed by PEAO, there were no differences in BP or MSNA between athletes and nonathlets. In contrast, a significant decrease in muscle pressor reflex responses was observed in the athletes during rhythmic exercises followed by PEAO. The possible relationship between this effect and changes in muscle energy supply, increased wash-out of metabolites, and reduced sensitivity of the muscle receptors in athletes is discussed.     

Effect of acute intestinal obstruction on erythrocyte membrane function

The amino acid composition of red blood cell membrane proteins had been studied in different stages of acute intestinal obstruction. Hydrophobic amino acids were revealed to increase and glutamate was found to decrease during the early period of acute intestinal obstruction. Later neutral amino acids and some of the main amino acids were stated to decrease. Shifts in the ratio of protein fractions seen in red blood cell membrane of rats with acute intestinal obstruction could be explained by changes followed in the amino acid composition. The data accumulated had demonstrated that such a significant modification of protein component of the red blood cell membrane could be one of the reasons of the erythrocyte membrane penetrability violation and could play the pathogenetic role in the occurrence of irreversibility changes in cases of the intestinal obstruction. All that was mentioned above had shown the necessity to use membrane protectors and antienzyme drugs in the postoperative period.     

Modulation of erythropoietin concentrations by manipulation of hypercarbia.

The present studies were done to determine whether preventing the respiratory alkalosis, which is known to occur with acute "hypoxic" stimuli, would lead to alterations in plasma concentrations of erythropoietin (Ep). Rats were subjected to two acute stresses, hypoxia and blood loss, separately and in combination, with and without the added stress of hypercarbia. Hypercarbia in all experimental groups was associated with a decrease in plasma concentrations of Ep. This reduction in plasma Ep with hypercarbia could not be fully explained by the higher arterial pO2S or p50S of the hypercarbic rats. Hypercarbia may have indirectly suppressed Ep production by increasing blood flow to the site of Ep production. Alternatively, the cell of origin of Ep could be sensitive to changes in pH and/or PCO2. It was further demonstrated that neither the onset nor the degree of reticulocytosis could be predicted by the plasma Ep concentrations. It is likely that the removal of red blood cells led to a decrease in marrow transit time with the early emergence of reticulocytes after acute blood loss.     

Kindlin-3 is essential for integrin activation and platelet aggregation

Integrin-mediated platelet adhesion and aggregation are essential for sealing injured blood vessels and preventing blood loss, and excessive platelet aggregation can initiate arterial thrombosis, causing heart attacks and stroke. To ensure that platelets aggregate only at injury sites, integrins on circulating platelets exist in a low-affinity state and shift to a high-affinity state (in a process known as integrin activation or priming) after contacting a wounded vessel. The shift is mediated through binding of the cytoskeletal protein Talin to the beta subunit cytoplasmic tail. Here we show that platelets lacking the adhesion plaque protein Kindlin-3 cannot activate integrins despite normal Talin expression. As a direct consequence, Kindlin-3 deficiency results in severe bleeding and resistance to arterial thrombosis. Mechanistically, Kindlin-3 can directly bind to regions of beta-integrin tails distinct from those of Talin and trigger integrin activation. We have therefore identified Kindlin-3 as a novel and essential element for platelet integrin activation in hemostasis and thrombosis.     

Nitrite infusion increases cerebral blood flow and decreases mean arterial blood pressure in rats: A role for red cell NO

It has been proposed that the reduction of nitrite by red cells producing NO plays a role in the regulation of vascular tone. This hypothesis was investigated in rats by measuring the effect of nitrite infusion on mean arterial blood pressure (MAP), cerebral blood flow (CBF) and cerebrovascular resistance (CVR) in conjunction with the accumulation of red cell NO. The relative magnitude of the effects on MAP and CBF as well as the time dependent changes during nitrite infusion are used to distinguish between the effects on the peripheral circulation and the effects on the cerebral circulation undergoing cerebral autoregulation. The nitrite infusion was found to reverse the 96% increase in MAP and the 13% decrease in CBF produced by L-NAME inhibition of e-NOS. At the same time there was a 20-fold increase in oxygen stable red cell NO. Correlations of the red cell NO for individual rats support a role for red cell nitrite reduction in regulating vascular tone in both the peripheral and the cerebral circulation. Furthermore, data obtained prior to treatment is consistent with a contribution of red cell reduced nitrite in regulating vascular tone even under normal conditions.     

Structural and Functional Characterization of Cleavage and Inactivation of Human Serine Protease Inhibitors by the Bacterial SPATE Protease EspPα from Enterohemorrhagic E. coli

EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins) by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI), α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition.