Dual antithrombotic therapy dose-dependently alters hemostatic plug structure and function.

BACKGROUND: Antithrombotic medications carry an inherent risk of bleeding, which may be exacerbated when anticoagulant and antiplatelet therapeutics are combined. Prior studies have shown different effects of antiplatelet vs anticoagulant drugs on the structure and function of hemostatic plugs in vivo. OBJECTIVES: We examined whether dual antithrombotic treatment consisting of combined antiplatelet and anticoagulant therapeutics alters hemostatic plug structure and function differently from treatment with either therapeutic alone. METHODS: Mice were treated with the P2Y12 antagonist clopidogrel and the factor Xa inhibitor rivaroxaban across a range of doses, either alone or in combination. The hemostatic response was assessed using a mouse jugular vein puncture injury model. Platelet accumulation and fibrin deposition were evaluated using quantitative multiphoton fluorescence microscopy, and bleeding times were recorded. RESULTS: Mice treated with clopidogrel alone exhibited a decrease in platelet accumulation at the site of injury, with prolonged bleeding times only at the highest doses of clopidogrel used. Mice treated with rivaroxaban alone instead showed a reduction in fibrin deposition with no impact on bleeding. Mice treated with both clopidogrel and rivaroxaban exhibited platelet and fibrin accumulation that was similar to that with either drug given alone; however, dual antithrombotic therapy resulted in impaired hemostasis at doses that had no impact on bleeding when given in isolation. CONCLUSION: Combined administration of antiplatelet and anticoagulant therapeutics exacerbates bleeding as compared to that with either drug alone, potentially via combined loss of both adenosine 5'-diphosphate- and thrombin-mediated platelet activation. These findings enhance our understanding of the bleeding risk associated with dual antithrombotic therapy.

Chemical, biological and protein-receptor binding profiling of Bauhinia scandens L. stems provide new insights into the management of pain, inflammation, pyrexia and thrombosis.

Bauhinia scandens L. (Family, Fabaceae) is a medicinal plant used for conventional and societal medication in Ayurveda. The present study has been conducted to screen the chemical, pharmacological and biochemical potentiality of the methanol extracts of B. scandens stems (MEBS) along with its related fractions including carbon tetrachloride (CTBS), di-chloromethane (DMBS) and n-butanol (BTBS). UPLC-QTOF-MS has been implemented to analyze the chemical compounds of the methanol extracts of Bauhinia scandens stems. Additionally, antinociceptive and anti-inflammatory effects were performed by following the acetic acid-induced writhing test and formalin-mediated paw licking test in the mice model. The antipyretic investigation was performed by Brewer Yeast induced pyrexia method. The clot lysis method was implemented to screen the thrombolytic activity in human serum. Besides, the in silico study was performed for the five selected chemical compounds of Bauhinia scandens, found by UPLC-QTOF-MS By using Discover Studio 2020, UCSF Chimera, PyRx autodock vina and online tools. The MEBS and its fractions exhibited remarkable inhibition in dose dependant manner in the antinociceptive and antiinflammatory investigations. The antipyretic results of MEBS and DMBS were close to the standard drug indomethacin. Investigation of the thrombolytic effect of MEBS, CTBS, DMBS, and BTBS revealed notable clot-lytic potentials. Besides, the phenolic compounds of the plant extracts revealed strong binding affinity to the COX-1, COX-2, mPGES-1 and plasminogen activator enzymes. To recapitulate, based on the research work, Bauhinia scandens L. stem and its phytochemicals can be considered as prospective wellsprings for novel drug development and discovery by future researchers.

Acute and Subchronic Toxicities and Safety Pharmacology Studies of a Bacillus Subtilisin in Dogs.

Subtilisin NAT, a Bacillus subtilisin, is widely applied as a functional food and considered to be one of the most exploitable potential oral thrombolytic agents. Subtilisin QK, another Bacillus subtilisin, is a serine protease fermented by Bacillus subtilis 02 and has a better thrombolytic effect. Therefore, subtilisin QK is typically used for evaluating the safety of Bacillus subtilisins. Here, we conduct several good laboratory practice (GLP)-compliant studies in non-rodent animal, i.e., in Beagle dogs, including acute toxicity, subchronic toxicity, and safety pharmacology studies. No adverse effects were evident in the acute and 28-d subchronic toxicity studies at doses up to 40000 FU/kg and 16000 FU/kg/d, respectively. In evaluating the pharmacological safety of up to 2000FU/kg subtilisin QK, we found no significant differences between the electrocardiograms, blood pressures, and respiration of beagle dogs. These findings suggest the safety of Bacillus subtilisin, providing reliable pharmacological and toxicological data for its development and popularization as a functional food and drug.

Sparganii Rhizoma: A review of traditional clinical application, processing, phytochemistry, pharmacology, and toxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Sparganii Rhizoma (SR), a traditional Chinese medicine (TCM), is the rhizome of Sparganium stoloniferum Buch.-Ham. mainly distributed in East Asia. It has been used for eliminating blood stasis, promoting the flow of Qi, removing the retention of undigested food and relieving pain in China for hundreds of years. AIM OF THE REVIEW: This review summarizes comprehensive information in traditional clinical application, processing, phytochemistry, pharmacology, quality control and toxicity of SR, in exploring future scientific and therapeutic potentials. MATERIALS AND METHODS: Pertinent information was systematically collected from several electronic scientific databases (e.g., Web of Science, PubMed, China Knowledge Resource Integrated, Springer, Elsevier, ScienceDirect, and Google Scholar), PhD and MS dissertations, and classic Chinese medical books. RESULTS: SR is a gynecological drug which is often used to treat dysmenorrhea, mass in the abdomen, amenorrhea due to blood stasis, and abdominal distension in TCM. Two kinds of processed products of SR are included in Chinese Pharmacopoeia, which have better pharmacological effects than the crude herb. Approximately 180 compounds have been identified from SR, including phenylpropanoids, flavonoids, anthraquinones, organic acids, alkaloids, steroids, volatile oils, diarylheptanes, etc. The crude extracts and isolated components of SR have been reported to have anti-tumor, antithrombotic, estrogen antagonistic , anti-inflammatory, analgesic, antioxidant, anti organ fibrosis and other pharmacological activities. SR also has reproductive toxicity. CONCLUSIONS: As an important TCM, SR has been demonstrated by modern pharmacological researches to have significant bioactivities, especially on anti-tumor, antithrombotic, and estrogen antagonistic activities. These activities provide prospects for the development of new drugs and therapeutics for future applications. Nevertheless, quality control and evaluation, in-depth pharmacological mechanism, and toxicological effect of SR require further detailed research.

YiQiFuMai Lyophilized Injection ameliorates tPA-induced hemorrhagic transformation by inhibiting cytoskeletal rearrangement associated with ROCK1 and NF-κB signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Thrombolytic therapy with tissue plasminogen activator (tPA) after ischemic stroke exacerbates blood-brain barrier (BBB) breakdown and leads to hemorrhagic transformation (HT). YiQiFuMai Lyophilized Injection (YQFM) is a modern preparation derived from Sheng-mai San (a traditional Chinese medicine). YQFM attenuates the BBB dysfunction induced by cerebral ischemia-reperfusion injury. However, whether YQFM can suppress tPA-induced HT remains unknown. AIM OF THE STUDY: We investigated the therapeutic effect of YQFM on tPA-induced HT and explored the underlying mechanisms in vivo and in vitro to improve the safety of tPA use against stroke. METHODS: Male C57BL/6J mice were subjected to 45 min of ischemia and 24 h of reperfusion. tPA (10 mg/kg) were infused 2 h after occlusion and YQFM (0.671 g/kg) was injected 2.5 h after occlusion. The in vitro effect of YQFM (100, 200, 400 µg/mL) on tPA (60 µg/mL)-induced dysfunction of the microvascular endothelial barrier in the brain following oxygen-glucose deprivation/reoxygenation (OGD/R) was observed in bEnd.3 cells. RESULTS: YQFM suppressed tPA-induced high hemoglobin level in the brain, mortality, neurologic severity score, BBB permeability, expression and activation of matrix metalloproteinase (MMP)-9 and MMP-2, and degradation of tight-junction proteins. Furthermore, YQFM significantly blocked tPA-induced brain microvascular endothelial permeability and phosphorylation of Rho-associated kinase (ROCK)1, myosin light chain (MLC), cofilin and p65 in vivo and in vitro. CONCLUSION: YQFM suppressed tPA-induced HT by inhibiting cytoskeletal rearrangement linked with ROCK-cofilin/MLC pathways and inhibiting the nuclear factor-kappa B pathway to ameliorate BBB damage caused by tPA.

Evaluation of carbon tetrachloride fraction of Actinodaphne angustifolia Nees (Lauraceae) leaf extract for antioxidant, cytotoxic, thrombolytic and antidiarrheal properties.

Actinodaphne angustifolia Nees (Family: Lauraceae) is commonly used in folk medicine against urinary disorder and diabetes. The objective of the present study was to evaluate the antioxidant, cytotoxic, thrombolytic, and antidiarrheal activities of carbon tetrachloride (CCl4) fraction of leaves of A. angustifolia (CTFAA) in different experimental models. Antioxidant activity was evaluated by using qualitative and quantitative assays, while antidiarrheal effects assessed with castor oil-induced diarrheal models in mice. The clot lysis and brine shrimp lethality bioassay were used to investigate the thrombolytic and cytotoxic activities, respectively. CTFAA showed antioxidant effects in all qualitative and quantitative procedures. The fraction produced dose-dependent and significant (P<0.05 and P<0.01) activities in castor oil-induced diarrheal models. Moreover, CTFAA significantly (P<0.05) demonstrated a 15.29% clot lysis effect in the thrombolytic test, and the brine shrimp lethality assay LC50 value was 424.16 µg/ml bioassay. In conclusion, the current study showed CTFAA has significant antidiarrheal effects along with modest antioxidant and thrombolytic effects, and these data warrant further experiment to justify and include CTFAA as a supplement to mitigate the onset of diarrheal and cardiovascular disease.

Characterization of active anticoagulant fraction and a fibrin(ogen)olytic serine protease from leaves of Clerodendrum colebrookianum, a traditional ethno-medicinal plant used to reduce hypertension.

ETHNOPHARMACOLOGICAL RELEVANCE: Cardiovascular diseases are the major cause of mortality and morbidity, causing over 17.9 million deaths a year worldwide. Currently used therapy is often having side effects and expensive, dietary interventions and alternative medicines are required. Clerodendrum colebrookianum has been used to treat cardiac hypertension but anticoagulant potency was not evaluated. AIM OF THE STUDY: To characterize an active anticoagulant fraction (AAFCC) and a 30 kDa fibrin(ogen)olytic serine protease (clerofibrase) isolated from aqueous leave extract of C. colebrookianum. MATERIALS AND METHODS: AAFCC/clerofibrase was subjected to extensive biochemical and pharmacological characterization including LC-MS/MS, amino acid compositional and GC-MS analyses. Interaction between clerofibrase with fibrinogen was studied by spectrofluorometric analysis. In vitro thrombolytic, antiplatelet and cytotoxicity assay were performed. In vivo toxicity, anticoagulant, defibrinogen and antithrombotic activities were determined on Swiss albino mice. RESULTS: The in vitro anticoagulant activity of AAFCC was found to be superior to heparin and clerofibrase and comparable to Nattokinase and warfarin. The proteomics and amino acid composition analyses suggest that clerofibrase is a previously uncharacterized novel plant protease capable of degrading the -αß chains of fibrinogen/fibrin. AAFCC/clerofibrase exerts their anticoagulant action via fibrinogenolytic activity and partially by antiplatelet activity albeit they have no effect on thrombin and FXa inhibition. The spectrofluorometric analysis revealed the binding of clerofibrase to fibrinogen but not to thrombin and FXa. The phytochemical constituents and bioactive components of AAFCC were characterized by biochemical, and GC-MS analyses. The AAFCC and clerofibrase inhibited collagen/ADP-induced mammalian platelet aggregation, showed in vitro thrombolytic activity, and non-cytotoxic to mammalian cells. The AAFCC showed and dose-dependent in vivo plasma defibrinogenating and anticoagulant activities and inhibited k-carrageen-induced thrombus formation in the tails of mice. CONCLUSION: The potent in vivo anticoagulant and antithrombotic effects of AAFCC suggests its pharmacological significance as herbal anticoagulant drug for the prevention and/or treatment of hyperfibrinogenemia- and thrombosis associated cardiovascular disorders.

Shear-sensitive nanocapsule drug release for site-specific inhibition of occlusive thrombus formation.

Essentials Vessel stenosis due to large thrombus formation increases local shear 1-2 orders of magnitude. High shear at stenotic sites was exploited to trigger eptifibatide release from nanocapsules. Local delivery of eptifibatide prevented vessel occlusion without increased tail bleeding times. Local nanocapsule delivery of eptifibatide may be safer than systemic antiplatelet therapies. SUMMARY: Background Myocardial infarction and stroke remain the leading causes of mortality and morbidity. The major limitation of current antiplatelet therapy is that the effective concentrations are limited because of bleeding complications. Targeted delivery of antiplatelet drug to sites of thrombosis would overcome these limitations. Objectives Here, we have exploited a key biomechanical feature specific to thrombosis, i.e. significantly increased blood shear stress resulting from a reduction in the lumen of the vessel, to achieve site-directed delivery of the clinically used antiplatelet agent eptifibatide by using shear-sensitive phosphatidylcholine (PC)-based nanocapsules. Methods PC-based nanocapsules (2.8 × 1012 ) with high-dose encapsulated eptifibatide were introduced into microfluidic blood perfusion assays and into in vivo models of thrombosis and tail bleeding. Results Shear-triggered nanocapsule delivery of eptifibatide inhibited in vitro thrombus formation selectively under stenotic and high shear flow conditions above a shear rate of 1000 s-1 while leaving thrombus formation under physiologic shear rates unaffected. Thrombosis was effectively prevented in in vivo models of vessel wall damage. Importantly, mice infused with shear-sensitive antiplatelet nanocapsules did not show prolonged bleeding times. Conclusions Targeted delivery of eptifibatide by shear-sensitive nanocapsules offers site-specific antiplatelet potential, and may form a basis for developing more potent and safer antiplatelet drugs.

Dodecafluoropentane Emulsion Extends Window for tPA Therapy in a Rabbit Stroke Model.

Dodecafluoropentane emulsion (DDFPe) nanodroplets are exceptional oxygen transporters and can protect ischemic brain in stroke models 24 h without reperfusion. Current stroke therapy usually fails to reach patients because of delays following stroke onset. We tested using DDFPe to extend the time window for tissue plasminogen activator (tPA). Longer treatment windows will allow more patients more complete stroke recovery. We test DDFPe to safely extend the time window for tPA thrombolysis to 9 h after stroke. With IACUC approval, randomized New Zealand white rabbits (3.4-4.7 kg, n = 30) received angiography and 4-mm blood clot in the internal carotid artery for flow-directed middle cerebral artery occlusion. Seven failed and were discarded. Groups were IV tPA (n = 11), DDFPe + tPA (n = 7), and no therapy controls (n = 5). DDFPe (0.3 ml/kg, 2 % emulsion) IV dosing began at 1 h and continued at 90 min intervals for 6 doses in one test group; the other received saline injections. Both got standard IV tPA (0.9 mg/kg) therapy starting 9 h post stroke. At 24 h, neurological assessment scores (NAS, 0-18) were determined. Following brain removal percent stroke volume (%SV) was measured. Outcomes were compared with Kruskal-Wallis analysis. For NAS, DDFPe + tPA was improved overall, p = 0.0015, and vs. tPA alone, p = 0.0052. For %SV, DDFPe + tPA was improved overall, p = 0.0003 and vs. tPA alone, p = 0.0018. NAS controls and tPA alone were not different but %SV was, p = 0.0078. With delayed reperfusion, DDFPe + tPA was more effective than tPA alone in preserving functioning brain after stroke. DDFPe significantly extends the time window for tPA therapy.

Toxicity studies of a bioactive protein with antithrombotic-thrombolytic activity, DLBS1033.

DLBS1033 is a bioactive protein extract containing Lumbricus rubellus and has been known to have antithrombotic/thrombolytic activity. The present study was aimed to assess the safety aspect of DLBS1033 in a preclinical setting, which included observation on toxic signs after acute and repeated administrations, and the drug's effect on prenatal development and drug interaction. In acute toxicity study, a high dose level (16.2 g/kg) of DLBS1033 was well tolerated. In subchronic toxicity study, after the doses of 270, 540 and 1080 mg/kg of DLBS1033 per day, no mortality was observed and other parameters were all observed to be normal. In prenatal developmental toxicity, no observed adverse effect level (NOAEL) of DLBS1033 was observed at a moderate dose (540 mg/kg). Coadministration of DLBS1033 with clopidogrel or aspirin did not cause gastric lesions, except when all three drugs were coadministrated. Taken together, results of the present study suggested that DLBS1033 is safe for long-term administration, with a caution at a high dose used during pregnancy, and can be used in combination with one of the antiplatelet drugs.

In vitro antioxidant, cytotoxic, thrombolytic activities and phytochemical evaluation of methanol extract of the A. philippense L. leaves.

OBJECTIVE: To study the leaves of Adiantum philippense L. for their antioxidant, cytotoxicity and thrombolytic activities and to perform phytochemical evaluation. METHODS: In-vitro antioxidant activity of extract was studied using DPPH radical scavenging, reducing power, total phenol and total flavonoid content determination assays. The cytotoxic activity was determined using brine shrimp lethality bioassay, thrombolytic activity by clot disruption and phytochemical potential by qualitative analysis. RESULTS: The antioxidant activity of the extracts was found promising. The reducing power of this crude extract increase with the increase of concentration; IC50 values of DPPH scavenging activity was (140.00±0.86) µg/mL as compared to ascorbic acid [IC50 (130.00±0.76) µg/mL]; Total phenol and total flavonoids content were (148.26±0.24) mg/mL and (163.06±0.56) mg/mL respectively. In cytotoxicity assay the LC50 values of the sample was (106.41±0.78) µg/mL where as for standard vincristin sulphate was (08.50±0.24) µg/mL as a positive control and the extract shows (12.86±1.02)% clot lytic whereas standard streptokinase shows (30.86±0.44% clot lytic activity in thrombolytic assay. The phytochemical evaluation indicates the presence of chemical constituents including carbohydrates, alkaloids, saponins, glycosides, flavonoids. CONCLUSIONS: This study shows that the methanol extract of leaves of Adiantum philippense L. has bioactivity but further compound isolation is necessary to confirm the activities of individual compounds.

ADAMTS13 exerts a thrombolytic effect in microcirculation.

Recombinant tissue plasminogen activator (r-tPA) is the drug of choice for thrombolysis, but it is associated with a significant risk of bleeding and is not always successful. By cleaving von Willebrand factor (VWF), the metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats-13) down-regulates thrombus formation in injured vessels. We investigated whether recombinant ADAMTS13 (r-ADAMTS13) induces thrombolysis in vivo in mice. Thrombosis was produced by ferric chloride-induced (FeCl(3)) injury in the venules of a dorsal skinfold chamber. Phosphate-buffered saline (PBS, vehicle), r-tPA or r-ADAMTS13, supplemented with hirudin (to stop on-going thrombin generation), was directly applied onto the occluded vessel, and thrombus dissolution was evaluated by intravital microscopy. The incidence of blood flow restoration significantly increased 30 minutes (min) after r-ADAMTS13 vs. PBS treatment (60% vs. 0%, p<0.05) and 60 min after r-tPA treatment (75% vs. 17%, p<0.05). Both r-tPA and r-ADAMTS13 significantly reduced thrombus size 60 min after their superfusion (53.2% and 62.3% of the initial thrombus size, p<0.05 and p<0.01, respectively). Bleeding occurred in all r-tPA-treated chambers, while it was absent in mice treated with r-ADAMTS13 or PBS. We observed that, similar to r-tPA, r-ADAMTS13 can dissolve occlusive thrombi induced by FeCl(3) injury in venules. In contrast to r-tPA, the in vivo thrombolytic effect of ADAMTS13 was not associated with any signs of haemorrhage. ADAMTS13 could represent a new therapeutic option for thrombolysis.

Protective effect of schizolobium parahyba flavonoids against snake venoms and isolated toxins.

Four compounds (isoquercitrin, myricetin-3-O-glucoside, catechin and gallocatechin) were isolated from lyophilized aqueous extract of Schizolobium parahyba leaves by chromatography on Sephadex LH-20, followed by semipreparative HPLC using a C-18 column, and identified by 1H and 13C NMR. The compounds were then, tested against hemorrhagic and fibrinogenolytic activities of Bothrops crude venoms and isolated metalloproteinases. The inhibitors neutralized the biological and enzymatic activities of Bothrops venoms and toxins isolated from B. jararacussu and B. neuwiedi venoms. The results showed that gallocatechin and myricetin-3-O-glucoside are good inhibitors of hemorrhagic and fibrinogenolytic activities of metalloproteinases, respectively. Gallocatechin also inhibited the myotoxic activity of both B. alternatus venom and BnSP-6 (Lys49 PhospholipaseA2 from B. neuwiedi). Circular dichroism and docking simulation studies were performed in order to investigate the possible interaction between BnSP-6 and gallocatechin. This is the first time these compounds and their anti-ophidian properties are reported for S. parahyba species. Forthcoming studies involving X-ray co-crystallization, will be of great importance for the development of new therapeutic agents for the treatment of ophidian accidents and for the better understanding of the structure/function relationship of venom toxins.

Ent-16ß,17-dihydroxy-kauran-19-oic acid, a kaurane diterpene acid from Siegesbeckia pubescens, presents antiplatelet and antithrombotic effects in rats.

The antiplatelet and antithrombotic effects of ent-16ß,17-dihydroxy-kauran-19-oic acid (DDKA) isolated from Siegesbeckia pubescens were investigated with different methods both in vitro and in vivo. We tested the antithrombotic activity of DDKA in arterio-venous shunt model. The effects of DDKA on adenosine diphosphate (ADP)-, Thrombin-, Arachidonic acid-induced rat platelets aggregation were tested in vitro. We also assessed its bleeding side effect by measuring coagulation parameters after intravenous administration for 5 days and investigated the potential mechanisms underlying such activities. In vivo, DDKA significantly reduced thrombus weight in the model of arterio-venous shunt. Meanwhile, DDKA increased plasma cAMP level determined by radioimmunoassay in the same model. Notably, DDKA prolonged PT and APTT in rats after intravenous administration DDKA for successive 5 days. In vitro, pretreatment with DDKA on washed rat platelets significantly inhibited various agonists stimulated platelet aggregation and caused an increase in cAMP level in platelets activated by ADP. These findings support our hypothesis that DDKA possesses antiplatelet and antithrombotic activities. The mechanisms underlying such activities may involve the anticoagulatory effect and cAMP induction.

Evaluating the possible genotoxic, mutagenic and tumor cell proliferation-inhibition effects of a non-anticoagulant, but antithrombotic algal heterofucan.

Fucan is a term used to denominate a family of sulfated polysaccharides rich in L-fucose. They are extracted mainly from brown seaweeds and echinoderms. The brown seaweed Spatoglossum schröederi (Dictyotaceae) synthesizes three heterofucans named A, B and C. Our research group purified a non-anticoagulant heterofucan (fucan A) which displays antithrombotic activity in vivo. However, its in vitro toxicity has yet to be determined. This work presents the evaluation of the potential cytotoxicity, mutagenicity and genotoxicity of this fucan. After 48 h incubation fucan A cytotoxicity was determinate using MTT assay. Tumor-cell (HeLa, PC3, PANC, HL60) proliferation was inhibited 2.0-43.7%; at 0.05-1 mg ml⁻¹ of the heterofucan, the 3T3 non-tumor cell line proliferation was also inhibited (3.3-22.0%). On the other hand, the CHO tumorigenic and RAW non-tumor cell lines proliferation were not affected by this molecule (0.05-1 mg ml⁻¹). We observed no mutagenic activity in Salmonella reversion assay when bacterial strains TA97a, TA98, TA100 and TA102 (with and without S9) were used.Comet assay showed that fucan A had no genotoxic effect (from 20 to 1000 mg ml⁻¹) on CHO cells. In conclusion, this study indicates that the S. schröederi fucan A was not found to be genotoxic or mutagenic compound; thus it could be used in new antithrombotic drug development.

Effects of polysaccharides enriched in 2,4-disulfated fucose units on coagulation, thrombosis and bleeding. Practical and conceptual implications.

Sulfated polysaccharides from marine invertebrates have well-defined structures and constitute a reliable class of molecules for structure-activity relationship studies. We tested the effects of two of these polysaccharides, namely a sulfated fucan and a fucosylated chondroitin sulfate, on coagulation, thrombosis and bleeding. The compounds share similar 2,4-disulfated fucose units, which are required for high anticoagulant activity in this class of polymer. These residues occur either as branches in fucosylated chondroitin sulfate or as components of the linear chain in the sulfated fucan. These polysaccharides possess anticoagulant activity but differ significantly in their mechanisms of action. The fucosylated chondroitin sulfate inhibits thrombin by heparin cofactor II, whereas sulfated fucan inhibits thrombin by both antithrombin and heparin cofactor II. In addition, these polysaccharides also have serpin-independent anticoagulant activities. Fucosylated chondroitin sulfate, but not sulfated fucan, activates factor XII. As a result of the complex anticoagulant mechanism, the invertebrate polysaccharides differ in their effects on experimental thrombosis. For instance, the sulfated fucan inhibits venous thrombosis at lower doses than fucosylated chondroitin sulfate. In contrast, fucosylated chondroitin sulfate is significantly more potent than sulfated fucan in arterial thrombosis. Finally, fucosylated chondroitin sulfate increases bleeding, while sulfated fucan has only a discrete effect. In conclusion, the location of 2,4-disulfated fucose units in the polysaccharide chains dictates the effects on coagulation, thrombosis and bleeding.

Influence of molecular weight of chemically sulfated citrus pectin fractions on their antithrombotic and bleeding effects.

Evaluated were the anticoagulant and antithrombotic activities, and bleeding effect of two chemically sulfated polysaccharides, obtained from citric pectin, with different average molar masses. Both low-molecular-weight (Pec-LWS, 3,600 g/mol) and high-molecular-weight sulfated pectins (Pec-HWS, 12,000 g/mol) had essentially the same structure, consisting of a (1-->4)-linked alpha-D-GalpA chain with almost all its HO-2 and HO-3 groups substituted by sulfate. Both polysaccharides had anticoagulant activity in vitro, although Pec-HWS was a more potent antithrombotic agent in vivo, giving rise to total inhibition of venous thrombosis at a dose of 3.5 mg/kg body weight. Surprisingly, in contrast with heparin, Pec-HWS and Pec-LWS are able to directly inhibit alpha-thrombin and factor Xa by a mechanism independent of antithrombin (AT) and/or heparin co-factor II (HCII). Moreover, Pec-HWS provided a lower risk of bleeding than heparin at a dose of 100% effectiveness against venous thrombosis, indicating it to be a promising antithrombotic agent.

Anti-glycoprotein VI treatment severely compromises hemostasis in mice with reduced alpha2beta1 levels or concomitant aspirin therapy.

BACKGROUND: Platelet inhibition is a major strategy to prevent arterial thrombosis, but it is frequently associated with increased bleeding because of impaired primary hemostasis. The activating platelet collagen receptor, glycoprotein VI (GP VI), may serve as a powerful antithrombotic target because its inhibition or absence results in profound protection against arterial thrombosis but no major bleeding in mice. METHODS AND RESULTS: Mice lacking (-/-) or expressing half-levels (+/-) of the other major platelet collagen receptor, integrin alpha2beta1, were injected with the anti-GP VI antibody JAQ1 and analyzed on day 5. Anti-GP VI treatment resulted in a marked hemostatic defect in alpha2-/- or alpha2+/- mice, as shown by dramatically prolonged tail bleeding times. Platelet adhesion to collagen was studied in an ex vivo whole-blood perfusion system under high shear conditions. Weak integrin activation by thromboxane A2 (TxA2) receptor stimulation restored defective adhesion of anti-GP VI-treated wild-type but not alpha2-/- or alpha2+/- platelets to collagen. This process required the simultaneous activation of the G(q) and G13 signaling pathways, as demonstrated by use of the respective knockout strains. Conversely, inhibition of TxA2 production by aspirin severely compromised hemostasis in anti-GP VI-treated or GP VI/Fc receptor gamma-chain-deficient but not control mice. CONCLUSIONS: Anti-GP VI therapy may result in defective hemostasis in patients with reduced alpha2beta1 levels or concomitant aspirin therapy. These observations may have important implications for a potential use of anti-GP VI-based therapeutics in the prevention of cardiovascular disease.

Ethanol plus caffeine (caffeinol) for treatment of ischemic stroke: preclinical experience.

BACKGROUND AND PURPOSE: Ethanol and caffeine are 2 common psychoactive dietary components. We have recently shown that low-dose ethanol plus caffeine results in a 70% to 80% reduction of infarct volume after reversible common carotid/middle cerebral artery (CCA/MCA) occlusion in rats. The combination (caffeinol) was effective after either oral pretreatment or intravenous administration starting up to 2 hours after stroke onset. Ethanol alone aggravated ischemic damage, while caffeine alone was without effect. Daily caffeinol for 2 weeks before ischemia eliminated the neuroprotection seen with acute treatment (tolerance). The purpose of our present study was to further characterize the properties of caffeinol as a possible treatment for ischemic stroke. METHODS: The transient CCA/MCA occlusion model was used in all experiments. Five sets of experiments were conducted (1) to test the effectiveness of various doses of ethanol (0.2 to 0.65 g/kg) and caffeine (3 to 10 mg/kg) in the caffeinol mixture; (2) to test whether the neuroprotective dose of caffeinol can improve behavioral dysfunction; (3) to test whether chronic ethanol or caffeine before ischemia will affect efficacy of caffeinol treatment; (4) to test whether the protective effect of caffeinol can be improved by pairing it with 35 degrees C hypothermia; and (5) to test whether caffeinol affects frequency of hemorrhage after administration of recombinant tissue plasminogen activator (rtPA) in ischemic animals. RESULTS: Doses as low as 0.2 g/kg of ethanol and 6 mg/kg of caffeine in the caffeinol were effective in reducing cortical infarct volume and behavioral dysfunction after transient CCA/MCA occlusion. Daily exposure to ethanol but not caffeine before CCA/MCA occlusion eliminated the therapeutic efficacy of acute caffeinol treatment, similar to the tolerance observed after chronic exposure to caffeinol. The therapeutic effect of caffeinol could be further improved by pairing it with mild intraischemic hypothermia, and caffeinol did not increase hemorrhagic infarction when given in combination with rtPA. CONCLUSIONS: Low doses of caffeinol, equivalent to no more than 2 to 3 cups of strong coffee and 1 cocktail, are consistently and highly neuroprotective, are well tolerated, can be added to other therapies to increase the effect of each, and do not interfere with or complicate rtPA therapy. Caffeinol is an appropriate candidate for clinical trial in stroke patients, although it may be less effective in patients with regular alcohol intake.

Distinct dose-dependent effects of plasmin and TPA on coagulation and hemorrhage.

All thrombolytic agents in current clinical usage are plasminogen activators. Although effective, plasminogen activators uniformly increase the risk of bleeding complications, especially intracranial hemorrhage, and no laboratory test is applicable to avoid such bleeding. We report results of a randomized, blinded, dose-ranging comparison of tissue-type plasminogen activator (TPA) with a direct-acting thrombolytic agent, plasmin, in an animal model of fibrinolytic hemorrhage. This study focuses on the role of plasma coagulation factors in hemostatic competence. Plasmin at 4-fold, 6-fold, and 8-fold the thrombolytic dose (1 mg/kg) induced a dose-dependent effect on coagulation, depleting antiplasmin activity completely, then degrading fibrinogen and factor VIII. However, even with complete consumption of antiplasmin and decreases in fibrinogen and factor VIII to 20% of initial activity, excessive bleeding did not occur. Bleeding occurred only at 8-fold the thrombolytic dose, on complete depletion of fibrinogen and factor VIII, manifest as prolonged primary bleeding, but with minimal effect on stable hemostatic sites. Although TPA had minimal effect on coagulation, hemostasis was disrupted in a dose-dependent manner, even at 25% of the thrombolytic dose (1 mg/kg), manifest as rebleeding from hemostatically stable ear puncture sites. Plasmin degrades plasma fibrinogen and factor VIII in a dose-dependent manner, but it does not disrupt hemostasis until clotting factors are completely depleted, at an 8-fold higher dose than is needed for thrombolysis. Plasmin has a 6-fold margin of safety, in contrast with TPA, which causes hemorrhage at thrombolytic dosages.