Thrombus Formation In Arterioles Research Articles

LRRC8 complexes are adenosine nucleotide release channels regulating platelet activation and arterial thrombosis.

Platelet shape and volume changes are early mechanical events contributing to platelet activation and thrombosis. Here, we identify single-nucleotide polymorphisms in Leucine-Rich Repeat Containing 8 (LRRC8) protein subunits that form the Volume-Regulated Anion Channel (VRAC) which are independently associated with altered mean platelet volume. LRRC8A is required for functional VRAC in megakaryocytes (MKs) and regulates platelet volume, adhesion, and agonist-stimulated activation, aggregation, ATP secretion and calcium mobilization. MK-specific LRRC8A cKO mice have reduced arteriolar thrombus formation and prolonged arterial thrombosis without affecting bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced ATP/ADP release to amplify agonist-stimulated calcium and PI3K-AKT signaling via P2X1, P2Y 1 and P2Y 12 receptors. Small-molecule LRRC8 channel inhibitors recapitulate defects observed in LRRC8A-null platelets in vitro and in vivo . These studies identify the mechanoresponsive LRRC8 channel complex as an ATP/ADP release channel in platelets which regulates platelet function and thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

A critical role for both CD40 and VLA5 in angiotensin II-mediated thrombosis and inflammation.

Angiotensin II (Ang-II)-induced hypertension is associated with accelerated thrombus formation in arterioles and leukocyte recruitment in venules. The mechanisms that underlie the prothrombotic and proinflammatory responses to chronic Ang-II administration remain poorly understood. We evaluated the role of CD40/CD40 ligand (CD40L) signaling in Ang-II-mediated microvascular responses and assessed whether and how soluble CD40L (sCD40L) contributes to this response. Intravital video microscopy was performed to analyze leukocyte recruitment and dihydrorhodamine-123 oxidation in postcapillary venules. Thrombus formation in cremaster muscle arterioles was induced by using the light/dye endothelial cell injury model. Wild-type (WT), CD40-/-, and CD40L-/- mice received Ang-II for 14 d via osmotic minipumps. Some mice were treated with either recombinant sCD40L or the VLA5 (very late antigen 5; α5β1) antagonist, ATN-161. Our results demonstrate that CD40-/-, CD40L-/-, and WT mice that were treated with ATN-161 were protected against the thrombotic and inflammatory effects of Ang-II infusion. Infusion of sCD40L into CD40-/- or CD40L-/- mice restored the prothrombotic effect of Ang-II infusion. Mice that were treated with ATN-161 and infused with sCD40L were protected against accelerated thrombosis. Collectively, these novel findings suggest that the mechanisms that underlie Ang-II-dependent thrombotic and inflammatory responses link to the signaling of CD40L via both CD40 and VLA5.-Senchenkova, E. Y., Russell, J., Vital, S. A., Yildirim, A., Orr, A. W., Granger, D. N., Gavins, F. N. E. A critical role for both CD40 and VLA5 in angiotensin II-mediated thrombosis and inflammation.

Carbon‐based nanomaterials accelerate arteriolar thrombus formation in the murine microcirculation independently of their shape

Although carbon-based nanomaterials (CBNs) have been shown to exert prothrombotic effects in microvessels, it is poorly understood whether CBNs also have the potential to interfere with the process of leukocyte-endothelial cell interactions and whether the shape of CBNs plays a role in these processes. Thus, the aim of this study was to compare the acute effects of two differently shaped CBNs, fiber-shaped single-walled carbon nanotubes (SWCNT) and spherical ultrafine carbon black (CB), on thrombus formation as well as on leukocyte-endothelial cell interactions and leukocyte transmigration in the murine microcirculation upon systemic administration in vivo. Systemic administration of both SWCNT and CB accelerated arteriolar thrombus formation at a dose of 1 mg kg(-1) body weight, whereas SWCNT exerted a prothrombotic effect also at a lower dose (0.1 mg kg(-1) body weight). In vitro, both CBNs induced P-selectin expression on human platelets and formation of platelet-granulocyte complexes. In contrast, injection of fiber-shaped SWCNT or of spherical CB did not induce leukocyte-endothelial cell interactions or leukocyte transmigration. In vitro, both CBNs slightly increased the expression of activation markers on human monocytes and granulocytes. These findings suggest that systemic administration of CBNs accelerates arteriolar thrombus formation independently of the CBNs' shape, but does not induce leukocyte-endothelial cell interactions or leukocyte transmigration.

Platelet Activation and Platelet-leukocyte Aggregation Elicited in Experimental Colitis Are Mediated by Interleukin-6

There is growing evidence for an interdependence of inflammation, coagulation, and thrombosis in acute and chronic inflammatory diseases. Inflammatory bowel diseases (IBD) are associated with a hypercoagulable state and an increased risk of thromboembolism. Although the IBD-associated prothrombogenic state has been linked to the inflammatory response, the mediators that link these 2 conditions remain unclear. Recent evidence suggests that interleukin-6 (IL-6) may be important in this regard. The objective of this study was to more fully define the contribution of IL-6 to the altered platelet function that occurs during experimental colitis. The number of immature and mature platelets, activated platelets, and platelet-leukocyte aggregates were measured in wild-type and IL-6 mice with dextran sodium sulfate (DSS)-induced colonic inflammation. DSS treatment of WT mice was associated with significant increases in the number of both immature and mature platelets, activated platelets, and platelet-leukocyte aggregates. These platelet responses to DSS were not observed in IL-6 mice. Chronic IL-6 infusion (through an Alzet pump) in WT mice reproduced all of the platelet abnormalities observed in DSS-colitic mice. IL-6-infused mice also exhibited an acceleration of thrombus formation in arterioles, similar to DSS. These findings implicate IL-6 in the platelet activation and enhanced platelet-leukocyte aggregate formation associated with experimental colitis, and support a role for this cytokine as a mediator of the enhanced thrombogenesis in IBD.

Alterations in the nitric oxide / soluble guanylyl cyclase pathway enhance the risk of myocardial infarction

Myocardial infarction (MI) results from sudden atherothrombotic occlusion of a coronary artery, exhibits a genetic predisposition and clusters in families. We performed whole exome sequencing and analysed identified targets. In a MI family, heterozygous mutations in two functionally related genes, GUCY1A3, encoding the α1‐subunit of the soluble guanylyl cyclase (α1‐sGC), and CCT7, encoding a chaperonin containing TCP‐1 complex (CCT) protein, segregate with the disease. All family members with the GUCY1A3 (p.Leu163Phefs*24) and the CCT7 mutation (p.Ser525Leu) were affected. Transfection of sGC in HEK cells yielded its expression and NO‐induced cGMP formation, but transfection of the GUCY1A3 mutation was ineffective. Downregulation of CCT7 in VSM decreased sGC expression verifying the importance of CCT7 herein. In platelets of affected probands α1‐sGC and NO‐induced cGMP formation was strongly reduced. Light‐dye induced thrombus formation in arterioles revealed the functional relevance of α1‐sGC. Time to occlusion was shorter in α1‐sGC deficient mice. Inhibition of endogenous NO accelerated thrombus formation only in wildtype and abrogated differences between genotypes indicating an absent inhibitory effect of NO in α1‐sGC−/− mice. In conclusion, dysfunctional NO/sGC signalling may lead to premature MI and α1‐sGC is decisive in continuous inhibition of thrombus formation thereby preventing MI.

The Endothelial Tyrosine Phosphatase SHP-1 Plays an Important Role for Vascular Haemostasis in TNFα-Induced InflammationIn Vivo

Introduction. Inflammation and endothelium-derived superoxides are important pathomechanisms in atherothrombotic diseases. We could previously show that the tyrosine phosphatase SHP-1 acts as a negative regulator in endothelial superoxide production. In this study we investigated the influence of SHP-1 on platelet-endothelium interaction and arterial thrombosis in TNFα-induced endothelial inflammation in vivo. Methods. Arteriolar thrombosis and platelet rolling in vivo were investigated in C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. Results. Inhibition of SHP-1 by the specific pharmacological inhibitor sodium stibogluconate did not significantly enhance platelet-endothelium interaction in vivo under physiological conditions but led to an augmented fraction of rolling platelets in TNFα-induced systemic inflammation. Accordingly, ferric-chloride-induced arteriolar thrombus formation, which was already increased by SHP-1 inhibition, was further enhanced in the setting of TNFα-induced inflammation. Platelet aggregation in vitro as well as ex vivo was not influenced by SHP-1-inhibition. In cultured endothelial cells, sodium stibogluconate increased TNFα-induced surface expression of p-selectin and von Willebrand factor. Additionally, TNFα increased SHP-1 activity and protein expression. Conclusions. The endothelial tyrosine phosphatase SHP-1 plays an important role for vascular hemostasis in vivo, which is crucial in TNFα-induced endothelial inflammation where it may serve as an autoinhibitory molecule to prevent excess inflammatory response and thrombus formation.

Abstract 215: Inflammation-Induced Thrombogenic Phenotype in the Microvasculature: The Roles of Enhanced Platelet Production and Reactivity

It is well recognized that different pathological conditions associated with inflammation are also accompanied by altered platelet function and enhanced thrombus development. Experimental colitis (induced by dextran sodium sulfate, DSS) and angiotensin II (AngII)-induced hypertension (HTN) are two examples of pathologically distinct inflammation models that exhibit enhanced platelet adhesion in venules and accelerated thrombus formation in arterioles. Whether these responses represent alterations in platelet production, life-span, and/or function remains unclear. In this study, we compared the changes in platelet production and reactivity (to thrombin) that occur in murine models of DSS colitis and AngII-induced HTN. Mature (TO-) and immature (TO+) platelets in blood were quantified using thiazole orange (TO) staining with flow-cytometry. The in vivo biotinylation method was used to determine the life-spans of TO+ and TO- platelets. Platelet aggregation velocity was measured using a low-angle light scattering technique. Thrombus formation, induced by light/dye injury, was monitored in cremaster muscle arterioles and platelet adhesion was quantified in the venules. Our findings indicate that both models of inflammation promote platelet adhesion and enhanced thrombus formation in the microvasculature. The enhanced thrombosis normally detected in both DSS-induced colitis and AngII-induced hypertension models was not evident in interleukin-6 (IL-6) deficient mice and in WT mice treated with an IL-6 receptor blocking antibody. DSS colitis was accompanied by an increased appearance of both TO+ and TO- platelets, increased platelet reactivity (aggregation) to thrombin, and no change in platelet turnover. The AngII HTN model exhibits an increase in platelet turnover, an increased number of TO- (but not TO+) platelets, and no change in platelet aggregation, suggesting that an altered endothelial cell phenotype likely makes a larger contribution to the accelerated arteriolar thrombosis in this model. The divergent responses in these two IL-6 dependent models of microvascular thrombosis may reflect the relative importance of platelets and microvascular endothelial cells as molecular targets for IL-6 mediated thrombogenic responses.

Mechanisms of enhanced thrombus formation in cerebral microvessels of mice expressing hemoglobin-S

AbstractThe microvasculature assumes an inflammatory and procoagulant state in a variety of different diseases, including sickle cell disease (SCD), which may contribute to the high incidence of ischemic stroke in these patients. This study provides evidence for accelerated thrombus formation in arterioles and venules in the cerebral vasculature of mice that express hemoglobin-S (βs mice). Enhanced microvascular thrombosis in βs mice was blunted by immunologic or genetic interventions that target tissue factor, endothelial protein C receptor, activated protein C, or thrombin. Platelets from βs mice also exhibited enhanced aggregation velocity after stimulation with thrombin but not ADP. Neutropenia also protected against the enhanced thrombosis response in βs mice. These results indicate that the cerebral microvasculature is rendered vulnerable to thrombus formation in βs mice via a neutrophil-dependent mechanism that is associated with an increased formation of and enhanced platelet sensitivity to thrombin.

Interleukin-1β Mediates the Extra-Intestinal Thrombosis Associated with Experimental Colitis

Inflammatory bowel diseases (IBDs) are associated with an increased risk for thromboembolism, which is often manifested as deep vein thrombosis or pulmonary embolism, at extra-intestinal sites. Although some of the cytokines that contribute to IBD pathogenesis are also known to alter the coagulation pathway, it remains unclear whether these mediators also contribute to the extra-intestinal thrombosis often associated with IBD. The objective of this study is to evaluate the role of interleukin (IL)-1β in enhanced extra-intestinal thrombosis observed in mice with dextran sodium sulfate (DSS)-induced colitis. IL-1β concentrations were measured in plasma, colon, and skeletal muscle of wild-type (WT) control and colitic mice. Microvascular thrombosis was induced in cremaster muscle microvessels by using a light/dye injury model. The effects of exogenous IL-1β on thrombus formation were determined in control WT mice. DSS-induced thrombogenesis was evaluated in WT mice treated with an IL-1β antibody and in IL-1 receptor-deficient (IL-1r(-/-)) mice. DSS-induced colonic inflammation in WT mice was associated with enhanced thrombus formation in arterioles. IL-1β concentrations were elevated in inflamed colon and skeletal muscle. Exogenous IL-1β enhanced thrombosis in control mice in a dose-dependent manner. DSS colitic mice treated with the IL-1β antibody as well as IL-1r(-/-) mice exhibited significantly blunted thrombogenic responses. These findings implicate IL-1β as a mediator of enhanced microvascular thromboses that occur in extra-intestinal tissues during colonic inflammation.

Arylazoamidoximes and Related Compounds as NO‐modulators

Three amidinoarylhydrazines 1, three arylazoamidines 2, and nine arylazoamidoximes 3 have been synthesized and investigated for their potential to function as nitric oxide (NO) modulators. In-vitro studies demonstrated that 2 and 3 inhibited platelet aggregation (2c, IC(50 )= 3 microM) which could also be shown in vivo by inhibition of thrombus formation in arterioles (3a, 22%). Moreover, for all compounds antihypertensive effects were examined in vivo with SHR rats, with 2a being the most potent candidate by lowering blood pressure by 19%. However, no common underlying mechanism of action could be shown. Some of these compounds released HNO non-enzymatically. Incubations with NO synthase isoforms (NOSs) revealed, that compounds 1 to 3 were weak substrates for NOSs but arylazoamidoximes 3 remarkably elevated the NOSs activity in the presence of L-arginine (3h, up to fivefold). In addition, we examined effects on arginase and dimethylarginine dimethylaminohydrolase (DDAH), two further enzymes involved in the complex regulation of NO biosynthesis, to elucidate whether the observed in-vivo effects can be traced back to their modulation. Furthermore, the metabolic fate of arylazoamidoximes 3 was addressed by investigation of a possible N-reductive biotransformation. In summary, novel NO-modulating compound classes are presented, among which arylazoamidoximes 3 are potent activators of NOS isoforms, and arylazoamidines 2 exert in-vivo effects by unknown mechanisms.

Key Role of Platelet Procoagulant Activity in Tissue Factor‐and Collagen‐Dependent Thrombus Formation in Arterioles and Venules In Vivo Differential Sensitivity to Thrombin Inhibition

Blood coagulation and platelet activation are mutually dependent processes, but contribute differently to venous and arterial thrombosis. We investigated the interplay of these processes in vivo in a mouse model of arteriolar and venular thrombus formation. Thrombus formation was studied by intravital (fluorescence) microscopy after topical application of FeCl3 on mouse mesenteric microvessels. Both in arterioles and venules, the thrombus-forming process relied on tissue factor-factor VII(a) interaction, collagen exposure, and glycoprotein VI-mediated platelet activation. Arterial thrombus formation was impaired by mild thrombin inhibition or platelet inhibition, while venous thrombosis was only suppressed by strong thrombin inhibition or by mild thrombin inhibition together with platelet inhibition. Phosphatidylserine-exposing platelets were present in thrombi of both vessel types, as detected with fluorescently labeled annexin A5. Injection of annexin A5 to shield exposed phosphatidylserine abolished thrombus formation in arterioles and venules, while mutant M1234-annexin A5 was ineffective. Arterial and venous thrombus formations were only slightly affected in mice carrying the factor V Leiden mutation, suggesting insensitivity to factor Va inactivation. In this microvascular model, the formation of both arterial and venous thrombi relies on collagen-induced platelet activation and tissue factor-induced thrombin generation. Activated, phosphatidylserine-exposing platelets play a key role in thrombus growth in arterioles and venules.

Role of the Protein C Pathway in the Extraintestinal Thrombosis Associated With Murine Colitis

Chronic inflammatory bowel diseases (IBD) are associated with an increased risk for thromboembolism. Although thrombosis is known to contribute to the morbidity and mortality of patients with IBD, the underlying mechanisms that contribute to the genesis of a hypercoagulable state during intestinal inflammation remain poorly defined. The objective of this study was to determine whether the protein C pathway contributes to the enhanced extraintestinal thrombosis that is associated with dextran sodium sulfate (DSS)-induced colitis in mice. Microvascular thrombosis was induced in cremaster muscle microvessels of normal and colitic mice using a light/dye injury model. DSS colitis enhanced thrombus formation in cremaster arterioles of wild-type mice. The DSS-induced thrombosis response was greatly attenuated in transgenic mice over expressing the endothelial protein C receptor. Activated protein C (APC), administered to colitic WT mice immediately prior to photoactivation, also afforded protection against thrombosis, and an anti-APC antibody enhanced thrombus formation. These findings indicate that elevated APC levels, derived from either endogenous or exogenous sources, confer protection against the extraintestinal thrombosis that accompanies colonic inflammation.

T1912 Calcineurine Inhibitors Accelerate Microvascular Thrombus Formation In Vivo

Thrombotic microangiopathy (TMA) is a fatal complication that occurs after combined pancreaskidney transplantation leading to allograft dysfunction and graft loss. Immunosuppressive drugs, such as calcineurin inhibitors, have been implicated in the development of nonimmune TMA. Tacrolimus has been shown to be associated with decreased NO-production, thus contributing to thrombus formation. ADMA acts as an endogenous NO-synthase inhibitor and is considered to be a marker of endothelial dysfunction. Aim of this study was to analyse the influence of different immunosuppressants on microvascular thrombus formation in vivo and to further examine the underlying endothelial function. Using the skin fold chamber in C57BL/6J mice, microvascular thrombus formation was induced photochemically and was quantitatively analyzed by intravital fluorescence microscopy. Mice were treated with tacrolimus (TAC: 10 mg/kg/d; n = 7), cyclosporine (CYA: 5 mg/kg/d) or sirolimus (RAPA: 1,5 mg/kg/d ip) on 3 consecutive days. Control mice received NaCl 0,9 % (10 ml/kg/d ip; n = 5). Drug plasma levels were examined to ensure therapeutic doses. Additionally, the systemic circulation of sPand sE-selectin as well as ADMA plasma levels were measured by ELISA. Application of immunosuppressants produced clinically relevant plasma levels (TAC: 8 ± 2 ng/ml, CYA: 214 ± 16 ng/ml; RAPA: 9 ± 1 ng/ml). Microvascular thrombus formation was significantly accelerated in mice receiving TAC compared to control mice (arteriolar and venular occlusion: 251 ± 101s and 179 ± 27 s vs. control 818 ± 221 s and 749 ± 231 s; p < 0,05). Application of CYA significantly increased thrombus formation only in venules (276 ± 49s; p < 0,05 vs. control), whereas RAPA had no significant effect on thrombosis induction. Plasma concentrations of sP-Selectin and sE-selectin were slightly reduced after TAC and CYA application. ADMA-levels however were significantly increased in mice receiving TAC (1,28 ± 0,16 μmol/l vs. control: 0,74 ± 0,13 μmol/l; p < 0,05). The present results show that clinically relevant plasma levels of cyclosporine enhanced thrombus formation only in venules whereas tacrolimus significantly accelerates thrombus formation in arterioles and venules. This effect could be explained by increased ADMA-levels in plasma resulting in impaired NO-bioavailability and increased thrombogenicity, thus presenting a new molecular mechanism in the development of transplant-thrombosis.

Role of the protein C pathway in the extra‐intestinal thrombosis associated with inflammatory bowel disease

Inflammatory bowel diseases (IBD) are associated with an increased risk for thromboembolism. While thrombosis is known to contribute to the morbidity and mortality of patients with IBD, the underlying mechanisms that contribute to the genesis of a hypercoagulable state during intestinal inflammation remain poorly defined. The objective of this study was to determine whether the protein C pathway contributes to the enhanced extra‐intestinal thrombosis that is associated with dextran sodium sulfate (DSS)‐induced colitis in mice. Intravital microscopy was used to monitor thrombus formation in arterioles and venules of the cremaster muscle in colitic wild type (WT) and mutant mice that overexpress the endothelial cell protein C receptor (EPCR). Thrombus formation was induced using the light/dye endothelial injury model and quantified using the time of onset of the thrombus and time to blood flow cessation. Some WT mice were treated with either activated protein C (APC) or an APC neutralizing antibody (APC‐Ab). The DSS‐enhanced thrombosis response was greatly attenuated in the EPCR transgenic mice and in WT mice treated with APC immediately prior to photoactivation. Treatment with the APC‐Ab further promoted the DSS‐enhanced thrombosis response. These findings indicate that an altered protein C pathway contributes to the extra‐intestinal thrombosis that accompanies colonic inflammation. (P01 DK43785)

In vivo thrombus formation.

Thrombus formation, including platelet adhesion, activation, secretion and aggregation as well as tissue factor-initiated thrombin generation and fibrin formation, has been studied in the past using in vitro systems, often with isolated components. Given the complexity of hemostasis and thrombosis, many of the concepts that have been developed to explain these processes are being revisited by studying thrombus formation in live animals using intravital microscopy and genetically altered mice. Although much of the dogma that has evolved has been confirmed by in vivo studies of thrombus formation, there have also been conflicts between old concepts and new direct observations. In vivo studies of the initiation of thrombus formation, platelet accumulation and thrombin generation have provided evidence for the participation of novel proteins and identified new pathways and mechanisms.

Extra‐intestinal thrombosis in experimental colitis

Inflammatory bowel disease (IBD) is associated an increased risk for thromboembolism. While thrombosis is known to contribute to morbidity and mortality in IBD, it remains unclear whether this hypercoagulable state can be recapitulated in animal models of colitis. The objectives of this study were to determine whether murine colitis is associated with enhanced thrombus formation in an extra-intestinal vascular bed, and whether genetic overexpression of the endothelial cell protein C receptor (ECPR) offers protection against the thrombus promoting effects of colitis. Intravital microscopy was used to monitor thrombus formation in arterioles and venules of the cremaster muscle in wild type (WT) and ECPR-transgenic mice placed on 3 % dextran sodium sulfate (DSS) in drinking water for 6 days. Thrombus formation was induced using the light/dye endothelial injury model and quantified using the time of onset and completion (blood flow cessation) of the formed thrombus. Our results show that DSS colitis is also associated with an enhanced thrombotic response that is consistent with a pro-coagulant state; this effect was largely manifested in arterioles but not in venules. Overexpression of ECPR significantly delayed thrombus formation. These findings indicate that extra-intestinal thrombus formation is enhanced in experimental IBD and that ECPR affords protection against this response. (P01 DK43785)

Endogenous Arteriolar Fibrinolysis Requires Urokinase Plasminogen Activator and Leukocyte Urokinase Plasminogen Activator Receptor-1.

Background: In venous thrombi, urokinase associated with leukocytes activates plasminogen to initiate endogenous fibrinolysis. Leukocytes also interact with arteriolar thrombi.Objectives: We hypothesized that urokinase plasminogen activator (uPA) activity would also be delivered to arteriolar thrombi by leukocytes.Methods: Using established techniques of high-speed intravital fluorescence microscopy to observe the cremaster muscle of mice, we measured platelet and fibrin accumulation in thrombi generated by a laser injury to arterioles. The accumulation of platelets in a thrombus is quantitated by measuring the fluorescence from anti-CD41 antibody tagged with a fluorescent secondary antibody and the presence of fibrin is detected using a fluorescence-tagged anti-fibrin antibody (T2G1).Results: In C57BL/6 wild type mice, fibrin increased to a maximum at about three minutes after thrombus formation and then decreased, such that by eight minutes after thrombus formation fibrin is present at 34% of its maximal value. When wild type mice are pretreated with epsilon-aminocaproic acid (16 mg/kg IV), the decrease in thrombus fibrin content was less (to 54% of maximal 8 minutes after thrombus formation), implying that the fibrin loss is at least partially the result of plasmin activity. Thrombus fibrin accumulation in tissue-plasminogen activator deficient mice (Plat) was greater, peaking at 229% the amount found in wild type mice, but the amount of fibrin present at eight minutes after thrombus formation was not dissimilar from wild type mice (45% ± 5% v. 34% ± 3%, P=0.09). In uPA-deficient mice (Plau) and uPA-receptor-1-deficient mice (Plaur), the amount of maximal thrombus fibrin was 189% and 273% that of fibrin content in wild type mice. Also, although thrombus fibrin did decrease over time in Plau and Plaur mice, it decreased to only 51% and 48% of maximal (each were P < 0.005 v. wild type) at eight minutes after thrombus formation. When wild type leukocytes, isolated from blood by differential centrifugation and sucrose density gradient, were transfused into Plaur mice, thrombus fibrin loss was restored to wild type levels (38% of maximal at 8 minutes after thrombus formation, P=0.36 vs. wild type, P=0.016 vs. Plaur), implying that transfused wild type leukocytes, which express uPA-receptor-1, can rescue fibrinolysis in uPA-receptor-1-deficient mice.Conclusions: These results confirm that endogenous fibrinolytic activity in vivo occurs soon after arteriolar thrombus formation. The plasminogen activators that are required for this fibrinolysis to occur are uPA, and the uPA-receptor-1 on leukocytes. These findings are compatible with the hypothesis that leukocytes can deliver plasminogen activator activity to arteriolar thrombi to initiate endogenous fibrinolysis.

Glycoprotein VI–dependent and –independent pathways of thrombus formation in vivo

The role of the collagen receptor glycoprotein VI (GPVI) in arteriolar thrombus formation was studied in FcRgamma-null mice (FcRgamma(-/-)) lacking platelet surface GPVI. Thrombi were induced with severe or mild FeCl(3) injury. Collagen exposure was significantly delayed and diminished in mild compared with severe FeCl(3) injury. Times to initial thrombus formation and vessel occlusion were delayed in FcRgamma(-/-) compared with wild-type mice after severe injury. Platelet accumulation in wild-type mice was decreased after mild compared with severe injury. However, there was little difference between platelet accumulation after severe or mild injury in FcRgamma(-/-). These data indicate a significant role for GPVI in FeCl(3)-induced thrombus formation. Pretreatment of wild-type mice with lepirudin further impaired mild FeCl(3)-induced thrombus formation, demonstrating a role for thrombin. Laser-induced thrombus formation in wild-type and FcRgamma(-/-) was comparable. Collagen exposure to circulating blood was undetectable after laser injury. Normalized for thrombus size, thrombus-associated tissue factor was 5-fold higher in laser-induced thrombi than in severe FeCl(3)-induced thrombi. Thus, platelet activation by thrombin appears to be more important after laser injury than platelet activation by GPVI-collagen. It may thus be important when considering targets for antithrombotic therapy to use multiple animal models with diverse pathways to thrombus formation.

Antithrombin is as effective as heparin and hirudin to prevent formation of microvascular thrombosis in a murine model

A recently published post-hoc analysis of a trial using high-dose antithrombin (AT) in septic patients (KyberSept) revealed significant reduction of lethality when no concomitant heparin was administered, whereas patients with the combination of heparin and AT did not benefit in terms of survival. Therefore, it seems feasible to study the capability of AT in prevention of microvascular thrombus formation to avoid concomitant application of heparin and AT. Using fluorescence microscopy and a light/dye-injury mouse ear model, the kinetics of thrombus formation were analyzed quantitatively in vivo upon single iv bolus of saline (control), heparin (100 IU/kg), hirudin (1 mg/kg) or AT (25, 50, 100 or 250 IU/kg) (N = 7 animals per group each). In controls, light/dye-injury induced complete thrombotic occlusion in all arterioles and venules studied. Heparin and hirudin prevented thrombotic vessel occlusion in 62% and 43% of arterioles and 11% and 28% of venules. AT-250 was found to be more effective than heparin and hirudin, because thrombus formation was completely banned in all arterioles and venules. AT-100 and AT-50 were also capable of significantly blocking thrombus formation in both arterioles and venules. In blood vessels, which finally clogged, the time for development of complete vessel occlusion was delayed after heparin, hirudin and AT-25, but in particular after AT-50 and AT-100. In conclusion, AT-mediated antithrombotic activity has been characterized in a model of phototoxicity-induced microvascular thrombosis formation, demonstrating that AT delays and prevents thrombus formation in arterioles and venules at least comparably effective as heparin and hirudin.

Sustained hypothermia accelerates microvascular thrombus formation in mice

Cold is supposed to be associated with alterations in blood coagulation and a pronounced risk for thrombosis. We studied the effect of clinically encountered systemic hypothermia on microvascular thrombosis in vivo and in vitro. Ferric chloride-induced microvascular thrombus formation was analyzed in cremaster muscle preparations from hypothermic mice. Additionally, flow cytometry and Western blot analysis was used to evaluate the effect of hypothermia on platelet activation. To test whether preceding hypothermia predisposes for enhanced thrombosis, experiments were repeated after hypothermia and rewarming to 37 degrees C. Control animals revealed complete occlusion of arterioles and venules after 742 +/- 150 and 824 +/- 172 s, respectively. Systemic hypothermia of 34 degrees C accelerated thrombus formation in arterioles and venules (279 +/- 120 and 376 +/- 121 s; P < 0.05 vs. 37 degrees C). This was further pronounced after cooling to 31 degrees C (163 +/- 57 and 281 +/- 71 s; P < 0.05 vs. 37 degrees C). Magnitude of thrombin receptor activating peptide (TRAP)-induced platelet activation increased with decreasing temperatures, as shown by 1.8- and 3.0-fold increases in mean fluorescence after PAC-1 binding to glycoprotein (GP)IIb-IIIa and 1.6- and 2.9-fold increases of fibrinogen binding on incubation at 34 degrees C and 31 degrees C. Additionally, tyrosine-specific protein phosphorylation in platelets was increased at hypothermic temperatures. In rewarmed animals, kinetics of thrombus formation were comparable to those in normothermic controls. Concomitantly, spontaneous and TRAP-enhanced GPIIb-IIIa activation did not differ between rewarmed platelets and those maintained continuously at 37 degrees C. Moderate systemic hypothermia accelerates microvascular thrombosis, which might be mediated by increased GPIIb-IIIa activation on platelets but does not cause predisposition with increased risk for microvascular thrombus formation after rewarming.