Jozef S. Mruk

Does Antithrombotic Therapy Influence Residual Thrombus After Thrombolysis of Platelet-Rich Thrombus? Effects of Recombinant Hirudin, Heparin, or Aspirin

BACKGROUND Thrombolysis to normal flow in patients with acute myocardial infarction preserves left ventricular function and decreases mortality. Failure of early reperfusion, reocclusion, or residual thrombus may be due to concurrent activation of the platelet-coagulation system. Thus, we hypothesized that the best concomitant antithrombotic therapy (recombinant [r]-hirudin, heparin, or aspirin) will maximally accelerate thrombolysis by r-tissue-type plasminogen activator (rTPA) and reduce residual thrombus. METHODS AND RESULTS Occlusive thrombi were formed in the carotid arteries of 29 pigs (by balloon dilatation followed by endarterectomy at the site of injury-induced vasospasm) and matured for 30 minutes before rTPA was started, with or without antithrombotic therapy. Thrombolysis was assessed with the use of angiography and measurement of residual thrombus. Pigs were allocated to one of five treatments: placebo, rTPA, rTPA plus r-hirudin, rTPA plus heparin, or rTPA plus intravenous aspirin. No placebo-treated pig reperfused. Two of six animals treated with rTPA alone reperfused compared with seven of seven animals treated with rTPA plus r-hirudin (reperfusion time, 33 +/- 10 minutes), six of seven animals treated with rTPA plus heparin (reperfusion time, 110 +/- 31 minutes), and two of six animals with rTPA plus aspirin. The activated partial thromboplastin time was prolonged in only the rTPA plus r-hirudin group (25 +/- 0.1 times baseline) and the rTPA plus heparin group (5.3 +/- 0.2 times baseline). Residual 111In-platelet and 125I-fibrin(ogen) depositions were lower in the heparin-treated group and lowest in the r-hirudin-treated group (heparin versus hirudin, respectively; incidence of residual macroscopic thrombus was six of six animals versus two of seven [P = .01]; 125I-fibrin(ogen), 170 +/- 76 versus 48 +/- 6 x 10(6) molecules/cm2 [P = .02]; 111In-platelets, 47 +/- 15 versus 13 +/- 2 x 10(6)/cm2, P = .10). No pigs developed spontaneous bleeding. CONCLUSIONS Thrombin inhibition with heparin or r-hirudin significantly accelerated thrombolysis of occlusive platelet-rich thrombosis, but only the best antithrombotic therapy (r-hirudin) eliminated or nearly eliminated residual thrombus.

Flavone-8-Acetic Acid (Flavonoid) Profoundly Reduces Platelet-Dependent Thrombosis and Vasoconstriction After Deep Arterial Injury In Vivo

BACKGROUND Flavone-8-acetic acid (FAA; [Flavonoid]), an adjuvant antitumor drug, inhibits ristocetin-induced aggregation of human platelets. The effect of FAA on platelet-dependent thrombosis was studied in vivo in the porcine carotid artery after deep arterial injury by balloon angioplasty. METHODS AND RESULTS (111)In-labeled autologous platelet and (125)I-labeled porcine fibrin(ogen) deposition, and the incidence of macroscopic mural thrombosis onto deeply injured artery (tunica media) were compared in 20 pigs (40+/-1 kg [mean+/-SEM], body surface area=1.0+/-0.1 m(2)), randomized to FAA bolus (n=10) of 5.5g/m(2), followed by an infusion at 0.14g. m(-2). min(-1) or placebo (n=10). Vasoconstriction was measured immediately beyond the dilated segment using quantitative angiography. Platelet deposition (x10(6)/cm(2) of carotid artery) was reduced over 12-fold in pigs treated with FAA (13+/-3 versus 164+/-51, P=0.001) compared with placebo. Fibrin(ogen) deposition (x10(12) molecules/cm(2) of carotid artery) did not significantly differ in FAA-treated pigs versus placebo (40+/-8 versus 140+/-69, P=0.08). Large mural thrombi were present in 100% of placebo-treated pigs versus very small thrombi in 40% of FAA-treated pigs (P=0.005). Vasoconstriction was reduced from 46+/-6% in the placebo group to 15+/-3% in the FAA group (P<0.001). Plasma level of FAA before angioplasty was 515+/-23 microgram/mL. The activated partial thromboplastin time was unchanged. The bleeding time was >2SD above the normal mean in 4 of 5 treated pigs (increased from 157+/-29 to 522+/-123 s). CONCLUSIONS FAA markedly reduced platelet deposition, mural thrombi, and injury-induced vasoconstriction after deep arterial injury, suggesting that a major inhibition of platelet glycoprotein Ibalpha may be beneficial therapy.

Nucleotide-Binding Oligomerization Domain 2 Receptor Is Expressed in Platelets and Enhances Platelet Activation and Thrombosis

Background— Pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) is well investigated in immunity, but its expression and function in platelets has never been explored. Method and Results— Using reverse transcription polymerase chain reaction and Western blot, we show that both human and mouse platelets express NOD2, and its agonist muramyl dipeptide induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentrations of thrombin or collagen, and clot retraction, as well. These potentiating effects of muramyl dipeptide were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2 dependently. Using intravital microscopy, we found that muramyl dipeptide administration accelerated in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express receptor-interacting protein 2, and provided evidence suggesting that mitogen activated-protein kinase and nitric oxide/soluble guanylyl cyclase/cGMP/protein kinase G pathways downstream of receptor-interacting protein mediate the role of NOD2 in platelets. Finally, muramyl dipeptide stimulates proinflammatory cytokine interleukin-1&bgr; maturation and accumulation in human and mouse platelets NOD2 dependently. Conclusions— NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets that link thrombotic events to inflammation.Background Pattern recognition receptor NOD2 (nucleotide binding oligomerization domain 2) is well investigated in immunity, its expression and function in platelets has never been explored.

Tumor vascular disrupting agent 5,6‐dimethylxanthenone‐4‐acetic acid inhibits platelet activation and thrombosis via inhibition of thromboxane A2 signaling and phosphodiesterase

5,6‐Dimethylxanthenone‐4‐acetic acid (DMXAA) is a tumor vascular disrupting agent under clinical trials as an adjacent antitumor agent. DMXAA is structurally similar to flavone‐8‐acetic acid (FAA), an old tumor vascular disrupting agent with antiplatelet and antithrombotic effects. In contrast to FAA, which causes bleeding in tumor patients, no bleeding has been reported in patients receiving DMXAA. Whether DMXAA also affects platelet function is not clear.

Platelet factor XIII gene expression and embolic propensity in atrial fibrillation

Nearly 15% of patients with non-valvular atrial fibrillation (NVAF) have left atrial appendage thrombus (LAAT) by transesophageal echocardiography (TEE) and yet the annual stroke rate averages 5%. The aim of this study was to identify variables influencing embolic propensity of LAAT. Platelet RNA was extracted from platelet-rich regions within formalin-fixed, paraffin-embedded specimens obtained from NVAF patients during cardiac surgery (26 LAAT from 23 patients) or peripheral embolectomy (51 thrombi from 41 patients). Platelet RNA was also assessed from whole blood from 40 NVAF patients. Expression of six platelet-predominate genes: H2A histone family, A1 domain of factor XIII, integrin α₂bβ₃; glycoprotein IX, platelet factor 4, glycoprotein Ib, was performed using TaqMan MGB-probe based quantitative real-time polymerase chain reaction. Platelet factor XIII subunit A gene expression was significantly lower in embolised compared to non-embolised thrombi as determined by normalised cycle threshold values (4.0 ± 1.2 v 2.8 ± 1.8, p=0.02). Expression of other genes did not differ by embolic status. In conclusion, RNA extracted from formalin-fixed, paraffin-embedded platelet-rich tissues can be used for analysis of platelet-predominate gene expression. Variable factor XIII gene expression in thrombi generated during NVAF may in part explain the propensity to embolisation.

Impact of atrial fibrillation on platelet gene expression

Platelets retain cytoplasmic messenger RNA and are capable of protein biosynthesis. Several diseases are known to impact the platelet transcriptome but the effect of non‐valvular atrial fibrillation (NVAF) on platelet RNA transcript is essentially unknown. The aim of this study was to evaluate the impact of NVAF on platelet RNA transcript by measuring platelet genes expression in consecutive NVAF patients before and 3‐4 months after pulmonary vein isolation (PVI) and compared to normal sinus rhythm controls (NSR).

NOD2 Receptor is Expressed in Platelets and Enhances Platelet Activation and Thrombosis

Background— Pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) is well investigated in immunity, but its expression and function in platelets has never been explored. Method and Results— Using reverse transcription polymerase chain reaction and Western blot, we show that both human and mouse platelets express NOD2, and its agonist muramyl dipeptide induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentrations of thrombin or collagen, and clot retraction, as well. These potentiating effects of muramyl dipeptide were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2 dependently. Using intravital microscopy, we found that muramyl dipeptide administration accelerated in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express receptor-interacting protein 2, and provided evidence suggesting that mitogen activated-protein kinase and nitric oxide/soluble guanylyl cyclase/cGMP/protein kinase G pathways downstream of receptor-interacting protein mediate the role of NOD2 in platelets. Finally, muramyl dipeptide stimulates proinflammatory cytokine interleukin-1&bgr; maturation and accumulation in human and mouse platelets NOD2 dependently. Conclusions— NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets that link thrombotic events to inflammation.Background Pattern recognition receptor NOD2 (nucleotide binding oligomerization domain 2) is well investigated in immunity, its expression and function in platelets has never been explored.

Platelet-predominate gene expression and reticulated platelets in nonvalvular atrial fibrillation: Effect of pulmonary veins isolation

Reticulated platelet (RP) content is increased in nonvalvular atrial fibrillation (NVAF). The purpose of this study was to determine if platelet content, morphology, and RP proportion are modulated by platelet genes.

Nucleotide-Binding Oligomerization Domain 2 Receptor Is Expressed in Platelets and Enhances Platelet Activation and ThrombosisCLINICAL PERSPECTIVE

Background— Pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) is well investigated in immunity, but its expression and function in platelets has never been explored. Method and Results— Using reverse transcription polymerase chain reaction and Western blot, we show that both human and mouse platelets express NOD2, and its agonist muramyl dipeptide induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentrations of thrombin or collagen, and clot retraction, as well. These potentiating effects of muramyl dipeptide were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2 dependently. Using intravital microscopy, we found that muramyl dipeptide administration accelerated in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express receptor-interacting protein 2, and provided evidence suggesting that mitogen activated-protein kinase and nitric oxide/soluble guanylyl cyclase/cGMP/protein kinase G pathways downstream of receptor-interacting protein mediate the role of NOD2 in platelets. Finally, muramyl dipeptide stimulates proinflammatory cytokine interleukin-1&bgr; maturation and accumulation in human and mouse platelets NOD2 dependently. Conclusions— NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets that link thrombotic events to inflammation.Background Pattern recognition receptor NOD2 (nucleotide binding oligomerization domain 2) is well investigated in immunity, its expression and function in platelets has never been explored.