Facilitates Platelet Aggregation Research Articles

Multi-parametric thrombus profiling microfluidics detects intensified biomechanical thrombogenesis associated with hypertension and aging.

Arterial thrombosis is a leading cause of death and disability worldwide with no effective bioassay for clinical prediction. As a symbolic feature of arterial thrombosis, severe stenosis in the blood vessel creates a high-shear, high-gradient flow environment that facilitates platelet aggregation towards vessel occlusion. Here, we present a thrombus profiling assay that monitors the multi-dimensional attributes of thrombi forming in such biomechanical conditions. Using this assay, we demonstrate that different receptor-ligand interactions contribute distinctively to the composition and activation status of the thrombus. Our investigation into hypertensive and older individuals reveals intensified biomechanical thrombogenesis and multi-dimensional thrombus profile abnormalities, endorsing the diagnostic potential of the assay. Furthermore, we identify the hyperactivity of GPIbα-integrin αIIbβ3 mechanosensing axis as a molecular mechanism that contributes to hypertension-associated arterial thrombosis. By studying drug-disease interactions and inter-individual variability, our work reveals a need for personalized anti-thrombotic drug selection that accommodates each patient's pathological profile.

Multi-parametric thrombus profiling microfluidics detects intensified biomechanical thrombogenesis associated with hypertension and aging.

Arterial thrombosis, which represents a critical complication of cardiovascular diseases, is a leading cause of death and disability worldwide with no effective bioassay for clinical prediction. As a symbolic feature of arterial thrombosis, severe stenosis in the blood vessel creates a high-shear, high-gradient flow environment that effectively facilitates platelet aggregation towards vessel occlusion even with platelet amplification loops inhibited. However, no approach is currently available to comprehensively characterize the size, composition and platelet activation status of thrombi forming under this biorheological condition. Here, we present a thrombus profiling assay that monitors the multi-dimensional attributes of thrombi forming in conditions mimicking the physiological scenario of arterial thrombosis. Using this platform, we demonstrate that different receptor-ligand interactions contribute distinctively to the composition and activation status of the thrombus. Our investigation into hypertensive and older individuals reveals intensified biomechanical thrombogenesis and multi-dimensional thrombus profile abnormalities, demonstrating a direct contribution of mechanobiology to arterial thrombosis and endorsing the diagnostic potential of the assay. Furthermore, we identify the hyperactivity of GPIbα-integrin αIIbβ3 mechanosensing axis as a molecular mechanism that contributes to hypertension-associated arterial thrombosis. By studying the interactions between anti-thrombotic inhibitors and hypertension, and the inter-individual variability in personal thrombus profiles, our work reveals a critical need for personalized anti-thrombotic drug selection that accommodates each patient's pathological profile.

Identification of Novel Ways Disrupting Platelet-Fibrinogen Interactions

Background: Platelets play a central role in thrombosis and hemostasis and immunity through their interaction with fibrinogen. Fibrinogen is a plasma protein that facilitates thrombosis and inflammation. Fibrinogen is the ligand for the major platelet receptor aIIbb3 and TREM- Like Transcript-1 (TLT-1), which platelets release from their a-granules, upon platelet activation. TLT-1 binding fibrinogen was surprising since αIIbβ3, the most abundant platelet receptor, binds fibrinogen, facilitating platelet aggregation. Although TLT-1 may assist in clot formation and hemostasis with αIIbβ3, TLT-1's main association is with regulating inflammatory-derived bleeding. Little is known about the TLT-1-fibrinogen interaction. The goal of this study is to identify the binding sites for this interaction and determine whether it can regulate inflammatory hemostasis in an LPS inflammatory mouse model. Aims: Delineate the TLT-1-fibrinogen binding site and determine if, peptides representing the molecular interaction affect platelet function. Methods: Using an Octet Qke Bio-layer Interferometry (BLI), we confirmed a binding interaction between TLT-1 and fibrinogen. Subsequently we digested fibrinogen using either trypsin or chymotrypsin and immunoprecipitated the peptides with a TLT-1-fc chimera followed by -Mass Spectrometry (LC-MS/MS) to identify potential interacting peptides. As an alternative method, we used a peptide microarray, where fluorescently labeled TLT-1 was incubated with 8-mers of α, β, γ chain fibrinogen peptides attached to a chip to determine which peptides bound TLT-1. Additionally, competitive ELISAs using peptides representing the TLT-1 extracellular domain loops (CDR-1, CDR2, CDR3 and LP17) were incubated with soluble TLT-1 in fibrinogen coated wells to determine the region of TLT-1 responsible for binding fibrinogen. To determine whether any of the selected peptides had inhibitory activity, we used two in vitro competitive inhibitory assays, first, we quantified FITC-labelled fibrinogen binding to activated human platelets and second, human platelet aggregometry incubated in the presence of these peptides at different concentrations (varying from 4-40 μM) using different agonists (ADP, thrombin, collagen, thromboxane A2). Results: We obtained an equilibrium dissociation constant (KD) of 3.02 ± 0.20 nM for the TLT-1 Fibrinogen interaction, suggesting a high affinity interaction. We identified four peptides that had consistent interaction without nonspecific binding in all three trials (trypsin and chymotrypsin IP, as well as the peptide microarray). The competitive platelet aggregometry and flow cytometry assays show decreased aggregation and significantly decreased fluorescent intensity of FITC-labelled fibrinogen in the presence of these peptides, suggesting two of these peptides are our potential binding sites (decrease of FITC-fluorescence intensity when compared to control: p1=20% decrease, p2=44% decrease, p3=21% decrease and P4=39% decrease, p=0.023). A HEK-293 transfected cell line expressing TLT-1 YFP is currently being used to access the ability of the identified peptides to mediate adhesion. In competitive ELISAs, TLT-1 loops LP17 and CDR3 show decreased absorbance when TLT-1 is incubated with fibrinogen in the presence of these peptides. Conclusions: Our TLT-1 data suggests that fibrinogen interacts with TLT-1 in the CD3 loop and in the region designated as LP17. With the aggregometry and flowcytometry competitive assays we have identified p2 as our most promising candidate for the TLT-1 interaction sight on fibrinogen. Currently we are adding the isolated peptides to our HEK/TLT-1 binding assay to determine if our selected peptide yields the same level of inhibition as seen in our aggregation and flow cytometric assays. We will then use an inflammatory in vivo mouse model to determine the effects of administering the selected peptide on platelet neutrophil conjugate formation and mortality. Our poster will report the current state of these studies .

Decreased level of TREM like Transcript 1 (TLT-1) is associated with prematurity and promotes the in-utero inflammatory response to maternal lipopolysaccharide (LPS) exposure.

The occurrence of preterm birth is associated with multiple factors including bleeding, infection and inflammation. Platelets are mediators of hemostasis and can modulate inflammation through interactions with leukocytes. TREM like Transcript 1 (TLT-1) is a type 1 single Ig domain receptor on activated platelets. In adults, it plays a protective role by dampening the inflammatory response and facilitating platelet aggregation at sites of vascular injury. TLT-1 is expressed in human placenta and found in cord blood. We thus hypothesized that TLT-1 deficiency is associated with prematurity and fetal inflammation. To test this hypothesis, we examined cord blood levels of soluble TLT-1 (sTLT) in premature and term infants and compared the inflammatory response in C57BL/6 (WT) and TLT-1-/- (treml1-/- , KO) mice given intraperitoneal LPS mid-gestation RESULTS: The preterm infant cord blood level of sTLT was significantly lower than that found at term. On exposure to LPS, histology of KO (as compared to WT) placenta and decidua showed increased hemorrhage, and KO decidual RNA expression of IL-10 was significantly lower. KO fetal interface tissues (placenta, membranes, amniotic fluid) over time showed increased expression of inflammatory cytokines such as IL-6, IFN-γ,and TNF, but not MCP-1. However, fetal organs showed similar levels. There is a potential association between insufficient TLT-1 expression and increased fetal inflammatory responses in the setting of prematurity. The data support further study of TLT-1 in the mechanistic link between bleeding, inflammation and preterm birth, and perhaps as a biomarker in human pregnancy.

TLT-1, a Potential Regulator of Inflammatory Pathogenesis in Obesity and Non-Alcoholic Fatty Liver Disease (NAFLD)

Background:Obesity, a nationwide health issue, has related medical costs ranging between $147-210 billion per year in United States and has been associated with a 3.5-fold increased risk of developing NAFLD. In obesity, platelets work in a pleotropic manner with vascular and immune cells to amplify the chronic inflammatory process. Interestingly, studies have demonstrated that platelet numbers and reactivity are increased in obese individuals. The emerging role of activated platelets during obesity induced inflammation introduces the novel concept of platelet targeted therapeutic interventions.Kopec et al, further supports the idea that the mechanism underlying the progression of obesity lies in a platelet mediated pro-inflammatory state, illustrating that there is extravascular fibrin(ogen) deposition, macrophages and inflammatory cytokines within white adipose tissue and liver of mice on western diet. Kopec et al uses a fibrinogen mutant mouse (Fiby390-396 ) which lacks the binding motif for Mac-1 and inhibits the ligand interaction with leukocytes, diminishing inflammation, reducing macrophage counts, reducing weight, protects mice from NAFLD and glucose dysmetabolism. Taken together, all evidence points towards a platelet/fibrinogen/leukocyte pathophysiological mechanism underlying the development of obesity.TREM-Like Transcript-1 (TLT-1) is a platelet specific receptor found in the a-granules of platelets and released to the surface upon platelet activation. TLT-1 is a type 1 receptor that, like the integrin a2bb3, binds fibrinogen and facilitates platelet aggregation . However, although TLT-1 may assist in clot formation and hemostasis to arrest bleeding in a non-inflammatory/nonimmune mediated setting, TLT-1's main association is with regulating inflammatory-derived bleeding. This is demonstrated by increased hemorrhage after inflammatory treatments such as lipopolysaccharide LPS in the treml1 -/- mice as compared to controls.Considering the emerging evidence in support of a platelet-fibrinogen receptor ligand interaction as a key mechanism underlying the development of obesity and that TLT-1, a platelet specific receptor binds fibrinogen and mediates leukocyte trafficking, our laboratory set out to determine whether TLT-1 could be implicated as the main culprit underlying this mechanism. When placed on a western diet, treml1 -/- mice are more prone to weight gain, based on these finding we hypothesize that: The TLT-1/Fibrinogen molecular interaction regulates metabolic inflammation in obesityAims:Evaluate the effects of western diet on obesity and NAFLD in the treml1 -/- mouse modelMethods:TLT-1 (treml1 -/-) - apolipoprotein E (apoe -/-) double null (AT-DKO;n=11) mice and control apoe +/-/treml1 +/- littermate controls (AT-Hets;n=20) were fed western diet for 20 weeks. Plasma samples were collected for adipokine, glucose, insulin, liver enzyme and lipid profiling. Mouse were perfused, liver and adipose tissue were collected for histological analysis.Results:Overall AT-DKO mice gained more weight compared to AT-Hets (12.94±1.90 vs 8.51±1.70 grams p=0.02). Plasma analysis demonstrates that the AT-DKO have higher levels of TNF-a (0.54±0.60 vs 0.118±0.17 pg/ml p=0.03), and IL-10 (2.50±1.40 vs 1.50±2.10 pg/ml p=0.004) compared to littermate controls. Histological analysis of livers illustrates increased lipid vacuoles and inflammatory foci in the AT-DKO mice as compared to controls, while preliminary data is not significant for these differences, liver damage in the AT-DKO was significantly greater as demonstrated by increased AST levels (166.21±91.00 vs 102±68.10 U/L p=0.02). Moreover, the AT-DKO mice had higher levels of ALT, direct bilirubin, cholesterol, pai-1 , triglycerides and lower IL-6 and Adiponectin (Table 1 data not significant). These findings suggest that in the absence of TLT-1 these mice are more prone to liver disfunction , hyperlipidemia and inflammatory alterations.Conclusions:Mutant AT-DKO mice are more prone to obesity and NAFLD compared to littermate controls, suggesting that TLT-1, a platelet gene, plays a surprising role in metabolism. Further investigation could adjudicate TLT-1 administration as a potential therapeutic intervention for prevention and amelioration of Obesity and related pathologies. The current state of this project will be reported here. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Deciphering the TLT-1 Fibrinogen Ligand Interaction

Background:Platelets, derived from megakaryocytes primarily play a central role in thrombosis and hemostasis, however, they extend beyond this role as immune cells that initiate and accelerate various vascular inflammatory conditions. Upon activation, platelets release TREM-Like Transcript-1 (TLT-1) from their a-granules onto their surface. Early studies by amino link columns preloaded with soluble TLT-1 followed by mass spectrometry and immunoblotting identified fibrinogen as a ligand for TLT-1. Fibrinogen is a plasma protein that is essential for clot formation, during inflammation and hypercoagulable states tissue deposition and plasma concentration of fibrinogen are increased, demonstrating a role of fibrinogen in both thrombosis and inflammation.TLT-1 binding fibrinogen was a surprising discovery since αIIbβ3, the most abundant platelet receptor, also binds fibrinogen and facilitates platelet aggregation. It is difficult to understand why there are two platelet specific receptors that have the same ligand, drawing us to question what the difference in function between the two is? Our studies suggest that although TLT-1 may assist in clot formation and hemostasis to arrest bleeding in a non-inflammatory setting like αIIbβ3, TLT-1's main association is with regulating inflammatory-derived bleeding. Very little is known about the TLT-1-Fibrinogen interaction, further studies would set the stage for a better understanding as to why two fibrinogen ligands exist on platelets and potentially outline a novel platelet therapeutic target during hypercoagulable and/or hyperinflammatory states. We set out to determine the binding affinity and localize the binding sites for the TLT-1 fibrinogen molecular interaction.Aims:Delineate the TLT-1 fibrinogen molecular interaction and elucidate the mechanism by which this interaction drives inflammation and thrombosis-hemostasis.Methods:To confirm the TLT-1 fibrinogen ligand interaction we carried out a kinetics assay using an Octet Qk e Bio-layer Interferometry (BLI) that measures biomolecular complex formation in real time. The TLT-1 Chimera was captured onto an Anti-Human Fc Capture (AHC) Biosensor, washed in kinetics buffer to limit nonspecific binding and submerged in a 96 well plate containing varying concentrations of Fibrinogen. To localize the exact binding sites for this molecular interaction, we digested fibrinogen using trypsin and carried out an immunoprecipitation (IP) followed by Liquid Chromatography-Mass Spectrometry (LC-MS/MS).Results:The curve (Figure 1) shows that the TLT-1 fibrinogen interaction has increasing bimolecular complex formation with increases in concentration of Fibrinogen (15.625nM - 250nM), with a concentration of 250nM showing the best bicomplex formation. In the control well with HIV01 4E10 capture, reference and sensor well, no bicomplex formation is shown, highlighting the specificity of the TLT-1 fibrinogen interaction. The curve illustrates a strong association with no dissociation, suggesting a strong interaction between the proteins. We isolated and identified four potential peptides (Alpha chain: GGSTSYGTGSETESPR, GSESGIFTNTK, Beta chain: QDGSVDFGR , QGFGNVATNTDGK) that bind TLT-1. We are currently performing BLI Competitive kinetics assays using biotinylated constructs of the peptides isolated from the Immunoprecipitation/ LC-MS/MS. The BLI competitive assays using the four peptides are suggestive of an interaction between TLT-1 and the four peptides as illustrated by increasing bimolecular complex formation with increasing concentration of soluble TLT-1 for all four peptides(data not shown).Conclusions:We obtained an equilibrium dissociation constant (KD) of 3.02 ± 0.20 nM for the TLT-1 fibrinogen interaction, suggesting a high affinity interaction between TLT-1 and fibrinogen. In our preliminary results from the BLI Competitive kinetics assays we obtained KD values within the nanomolar concentration range and are currently conducting experiments to optimize conditions to obtain our final bicomplex binding curve and KD values. We are currently assessing the identified peptides for potential of mediating the molecular interaction between TLT-1 and fibrinogen. Our poster will report the current state of these studies . [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Thrombosis and Von Willebrand Factor in Chuvash Polycythemia

Background: In Chuvash polycythemia (CP), homozygous germline VHLR200W results in augmented hypoxia-sensing, elevated erythropoietin and hemoglobin, and increased morbidity and mortality from venous and arterial thromboses.1 In CP patients and controls from Russia's Chuvash Republic, the probability of new thrombosis in CP increased with age, smoking, and higher expression of THBS1, the gene encoding thrombospondin-1.2 In mice, hypoxia leads to upregulation of THBS1 through HIF-23 and to upregulation of VWF .4 THBS1 reportedly regulates the size of circulating von Willebrand Factor (VWF) multimers5 through inhibiting proteolytic cleavage of VWF by ADAMTS13.6 We hypothesized that upregulation of THBS1 in CP may be a risk for thrombosis through an effect on ADAMTS13 and VWF.Methods: We enrolled into an observational study 155 patients with CP and 154 controls matched by age, sex and place of residence. Written informed consent was obtained from all subjects according to the Declaration of Helsinki. We genotyped for VHLR200W, measured baseline plasma THBS-1 concentrations2 and followed the subjects for a median of 9.4 years. Here, we report results for determining baseline circulating levels of ADAMTS13 antigen, activity and specific activity and of VWF antigen and differential multimers in up to 57 CP patients and 30 controls.Results: CP subjects had higher hemoglobin and erythropoietin concentrations and lower white blood cell and platelet counts (Table 1). They were also more likely to be current smokers. There was a trend to higher THBS-1 concentrations in the CP patients (p=0.084), which was significant after adjusting for platelet count (P=0.016). ADAMTS13 and VWF antigen levels did not differ between CP patients and controls but ADAMTS13 activity and especially specific activity were lower in CP. Plasma THBS-1 concentration correlated positively with ADAMTS13 antigen concentration (r=0.358, P=0.001) but negatively with specific activity (r=-0.363, P=0.001) (Figure 1). Eight (14.0%) CP patients (aged 27-76) years had a history of thrombosis (Table 1). The mean serum VWF antigen concentration (P=0.001) and differential VWF multimer size (P=0.090) were higher in CP patients with a history of thrombosis (Figure 2). During the prospective follow-up period, 10 CP patients (17.5%) experienced a new thrombosis at a median age of 46 (range 26-70) years. Three patients also experienced an additional thrombosis for a total of 13 new thromboses: myocardial infarction (n=5), stroke (n=3), splanchnic thrombosis (n=2), deep vein thrombosis (n=2), pulmonary embolism (n=1). Baseline serum VWF antigen levels (P=0.019) and VWF multimer size (P=0.099) were higher in subjects who developed a new thrombosis during follow-up (Figure 3), and these relationships persisted after adjusting for smoking history.Discussion: THBS1 is an adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions and facilitates platelet aggregation.7 In mice, platelet-derived THBS1 modulated arterial thrombosis only in the presence of VWF.8 Our present findings suggest that CP subjects have lower ADAMTS13 specific activity than controls and that this lower activity may be related to higher plasma THBS1 concentrations. Lower ADAMTS13 specific activity suggests the presence of an inhibitor or partial inactivation of the protease. Furthermore, higher VWF antigen and multimer size in CP patients is associated with a past history of thrombosis and a greater risk for new thrombosis in the future. More broadly, our findings suggest a possible implication of VWF in thrombosis of conditions associated with an up-regulated hypoxic response. Further studies may identify new pathways of prevention and treatment with broad applicability.

Highly oxidized low-density lipoprotein does not facilitate platelet aggregation.

ObjectiveThis study aimed to examine whether oxidized low-density lipoprotein (oxLDL) facilitates platelet aggregation, which is one cause for development of cardiovascular disease.MethodsThe susceptibility of platelets to aggregation was monitored by light transmittance aggregometry and a laser light scattering method using low-density lipoprotein (LDL) and oxLDL as agonists. β-thromboglobulin (β-TG) levels released from platelets were also measured after incubation with or without oxLDL.ResultsPlatelet aggregation was suppressed by oxLDL as estimated by maximum light transmission. Additionally, adenosine diphosphate-induced further aggregation was slightly reduced by the presence of oxLDL. Aggregation levels of a low number of platelets, which was determined by the laser light scattering method, were lower upon addition of oxLDL compared with unoxidized LDL. After a short time of incubation, oxLDL increased secreted β-TG levels in platelet-rich plasma. However, further incubation with oxLDL caused relatively lower secreted β-TG levels compared with incubation with unoxidized LDL. This fluctuation was not due to β-TG degradation by oxLDL.ConclusionsLevels of oxLDL in vitro weakly activate platelets at an early stage, but then inhibit platelet function, such as aggregation and β-TG secretion.

Regulatory Phenomena in the Glutathione Peroxidase Superfamily.

Significance: The selenium-containing Glutathione peroxidases (GPxs)1-4 protect against oxidative challenge, inhibit inflammation and oxidant-induced regulated cell death. Recent Advances: GPx1 and GPx4 dampen phosphorylation cascades predominantly via prevention of inactivation of phosphatases by H2O2 or lipid hydroperoxides. GPx2 regulates the balance between regeneration and apoptotic cell shedding in the intestine. It inhibits inflammation-induced carcinogenesis in the gut but promotes growth of established cancers. GPx3 deficiency facilitates platelet aggregation likely via disinhibition of thromboxane biosynthesis. It is also considered a tumor suppressor. GPx4 is expressed in three different forms. The cytosolic form proved to inhibit interleukin-1-driven nuclear factor κB activation and leukotriene biosynthesis. Moreover, it is a key regulator of ferroptosis, because it reduces hydroperoxy groups of complex lipids and silences lipoxygenases. By alternate substrate use, the nuclear form contributes to chromatin compaction. Mitochondrial GPx4 forms the mitochondrial sheath of spermatozoa and, thus, guarantees male fertility. Out of the less characterized GPxs, the cysteine-containing GPx7 and GPx8 are unique in contributing to oxidative protein folding in the endoplasmic reticulum by reacting with protein isomerase as an alternate substrate. A yeast 2-Cysteine glutathione peroxidase equipped with CP and CR was reported to sense H2O2 for inducing an adaptive response. Critical Issues: Most of the findings compiled are derived from tissue culture and/or animal studies only. Their impact on human physiology is sometimes questionable. Future Directions: The expression of individual GPxs and GPx-dependent regulatory phenomena are to be further investigated, in particular in respect to human health.

LPS enhances platelets aggregation via TLR4, which is related to mitochondria damage caused by intracellular ROS, but not extracellular ROS

Platelet is an important cell contributing to hemostasis and immunity. Bacterial lipopolysaccharide (LPS), mainly functioning by stimulating toll-like receptor 4 (TLR4), mediates platelet activation and sepsis. However, the inter-relationship between these players in sepsis remains unknown. We found that the aggregation of platelets was enhanced in complete blood of sepsis patients than that of healthy donors. PRP isolated from complete blood of healthy donors was used in the following study to filter out the interference of irrelevant cells. The results shown that the maximum aggregation rate (MAR) was significantly higher in LPS-challenged PRP model than that of controls, and administration of the specific TLR4 inhibitor, TAK242, reduced the MAR in this model. LPS promoted P-selectin expression and intracellular ROS production, and both TAK242 and N-acetyl-L-cysteine (NAC) could depressed the LPS-induced increase of P-selectin and intracellular ROS. H2O2 administration increased P-selectin expression partially but had little effect on intracellular ROS, thought it increased mitochondrial damage. In vivo, LPS increased both intracellular ROS and CD62P comparing with that of controls, effects that were prevented by TAK242. Furthermore, platelet aggregation through LPS-TLR4 pathway was involved in AKT, PKC and p38 phosphorylation but not cGMP/cAMP pathway. In conclusion, this study shows that intracellular ROS, not extracellular ROS such as H2O2, plays a crucial role in facilitating platelet aggregation via LPS/TLR4 pathway, and this process was involved in AKT, PKC and p38 phosphorylation but not cGMP/cAMP pathway. The results would helpful for understanding the role of intracellular ROS and LPS-TLR4 pathway in platelet aggregation.

Expression of matrix metalloproteinases-12 in ST-segment elevation myocardial infarction: A case-control study.

Matrix metalloproteinases-12 (MMP12) can lead to degradation of elastin resulting in plaque destabilization and rupture. MMP12 also facilitates platelet aggregation, adhesion, and granule secretion. However, evidence in the literature related to the function of MMP12 in ST-segment elevation myocardial infarction (STEMI) is little. This study investigated the expression of MMP12 in human coronary thrombus and examined the relationship between plasma MMP12 and STEMI.Arterial plasma was obtained from 46 STEMI patients and 52 stable angina pectoris (SAP) patients and 30 controls with angiographically normal coronary arteries. Coronary thrombi were obtained from 26 STEMI patients with a large thrombus burden (LTB). The expression levels of MMP12 in coronary thrombus were analyzed by immunohistochemistry and immunofluorescence, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting (WB) and casein zymography. In addition, MMP12 concentration measured by enzyme-linked immunosorbent assay (ELISA) and activity measured by fluorescence resonance energy transfer (FRET) were used to assess the levels in plasma.We confirmed the expression of MMP12 in human coronary thrombus. MMP12 was secreted mainly in active form of 45 kDa in coronary thrombus. In plasma samples of the STEMI group, MMP12 concentrations were found to be higher than the SAP group (5.030 ± 2.24 pg/mL vs 3.010 ± 1.99 pg/mL, P < .05) but with lower MMP12 activity (332 ± 77 RFU vs 458 ± 91 RFU, P < .05). Also, the STEMI group demonstrated much higher MMP12 concentrations than the normal coronary artery control group (5.030 ± 2.24 pg/mL vs 1.720 ± 0.51 pg/mL, P < .05) and with lower MMP12 activity (332 ± 77 RFU vs 549 ± 112 RFU, P < .05). In addition, the STEMI group had significantly higher tissue inhibitor of metalloproteinases-1 (TIMP1) concentration (573.40 ± 270.60 pg/mL) than SAP group (384.50 ± 147.70 pg/mL) and control group (219.90 ± 154.80 pg/mL, P < .05). The imbalance in MMP12/TIMP ratio was observed in the STEMI group compared with SAP and control group (P < .05).This study demonstrated that MMP12 exists in human coronary thrombus. Patients with STEMI have elevated plasma level of MMP12 and the imbalance of MMP12/TIMP1. These data supported that MMP12 might be of potential relevance in STEMI.

Thrombopoietin and platelet aggregation in patients with stable coronary artery disease

Thrombopoietin (TPO) may facilitate platelet activation and aggregation. However, data on the impact of TPO on platelet aggregation in patients with stable coronary artery disease (CAD) are scarce. We aimed to investigate associations between TPO and platelet aggregation and activation in patients with stable coronary artery disease (CAD). We studied 900 stable CAD patients. Serum TPO was assessed by ELISA. Platelet aggregation was evaluated using the Multiplate Analyzer (agonists: arachidonic acid [AA] and collagen) and the VerifyNow Aspirin Assay. Platelet activation was evaluated by soluble (s)P-selectin. Cyclooxygenase-1 inhibition was evaluated by serum thromboxane B2 (TXB2). We found that TPO correlated weakly with platelet aggregation evaluated by Multiplate using AA (r = −0.09, p = 0.01) and collagen as agonists (r = −0.03, p = 0.43) and by VerifyNow (r = 0.07, p = 0.03). We found no correlation between TPO and sP-selectin (r = −0.01, p = 0.70). Independent predictors of AA-induced platelet aggregation by Multiplate included high levels of sP-selectin and serum TXB2, high platelet count, increasing age and body mass index, female sex, and active smoking. Independent predictors of TPO included low AA-induced platelet aggregation by Multiplate, high levels of hs-CRP, active smoking, and high platelet aggregation evaluated by VerifyNow. In conclusion, TPO levels did not correlate with platelet activation and only weak associations were found between TPO and platelet aggregation, suggesting that TPO did not substantially facilitate platelet aggregation in stable CAD patients.

In silico Approach for Anti-Thrombosis Drug Discovery: P2Y1R Structure-Based TCMs Screening.

Cardiovascular diseases (CVDs), including thrombosis, which is induced by platelet aggregation, are the leading cause of mortality worldwide. The P2Y1 receptor (P2Y1R) facilitates platelet aggregation and is thus an important potential anti-thrombotic drug target. The P2Y1R protein structure contains a binding site for receptor antagonist MRS2500 within its seven-transmembrane bundle, which also provides suitable pockets for numerous other ligands to act as nucleotide antagonists of P2Y1R. The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) comprises 499 Chinese Pharmacopoeia-registered herbs and the structure information for 29,384 ingredients. In silico docking of these compounds into the P2Y1R protein structure within the MRS2500 pocket can identify potential antithrombotic drugs from natural medicinal plants. Docking studies were performed and scored to evaluate ligand-binding affinities. In this study, a total of 8987 compounds from Traditional Chinese Medicine (TCM) were filtered by Lipinski's rule of five, and their ideal oral-intake properties were evaluated. Of these, 1656 compounds distributed in 443 herbs docked into the P2Y1R-MRS2500 structure in 16,317 poses. A total of 38 compounds were ranked with a DockScore above 70, and these may have significant potential for development into anti-thrombosis drugs. These computational results suggested that licorice (Glycyrrhiza uralensis Fisch), cimicifugae (Cimicifuga foetida L.), and ganoderma (Ganoderma lucidum Karst) and their chemical constituents, which have not previously been widely used for anti-thrombosis, may have unexpected effects on platelet aggregation. Moreover, two types of triterpene scaffolds summarized from 10 compounds were distributed in these three herbs and also docked into P2Y1R. These scaffold structures may be utilized for the development of drugs to inhibit platelet aggregation.

Phosphatidylserine-Mediated Platelet Clearance By Endothelium Decreases Platelet Aggregates and Procoagulant Activity in Sepsis

Introduction: Disorders of coagulation are common in sepsis, with disseminated intravascular coagulation (DIC) occurring in approximately 35 % of severe cases, contributing to microvascular dysfunction and death. Intensive platelet activation in sepsis facilitates platelet aggregation, leading to the formation of microthrombi and platelet depletion. This results in the development of DIC and sepsis-associated thrombocytopenia. Therefore, platelets must be cleared locally and quickly in the early phase of activation. Previous studies mainly focused on the clearance of activated cold-stored and aging platelets as well as platelets in immune-mediated thrombocytopenia. However, platelet activation and their clearance in sepsis are poorly understood. Platelets can form aggregates with leukocytes resulting in leukocyte death, the release of extracellular traps (ETs), increased endothelial permeability, and aggravated thrombosis. This study explored an alternate pathway for platelet disposal mediated by endothelial cells (ECs) through phosphatidylserine (PS) and examined the effect of platelet clearance on procoagulant activity (PCA) in sepsis.Methods: The subjects were septic patients (n=48) and healthy controls (n=48). Platelet engulfment by ECs was observed by electron microscopy, immunofluorescence, or immunochemistry both in vitro and in animal models. The PCA of platelets was measured by clotting time, purified coagulation complex assays, and fibrin formation.Results: Platelets in septic patients demonstrated increased levels of surface activation markers and apoptotic vesicle formation, and also formed aggregates with leukocytes. Activated platelets adhered to and were ultimately digested by ECs in vivo and in vitro. Blocking PS on platelets or integrin on ECs attenuated platelet clearance, resulting in increased platelet count in a mouse model of sepsis (p<0.05). Furthermore, platelet removal by ECs resulted in a corresponding decrease in platelet-leukocyte complex formation and markedly reduced generation of factor Xa and thrombin on platelets (p<0.01). Pretreatment with lactadherin increased phagocytosis of platelets by approximately 2-fold, diminished PCA by 70%, prolonged coagulation time, and attenuated fibrin formation by 50%. A large decline in PS exposure on platelets, associated platelet PCA, and PLA formation is seen in patients in remission, which could be attributed to the elimination of abnormal platelets.Conclusions: Our results suggest that PS-mediated clearance of activated platelets by the endothelium results in an anti-inflammatory, anticoagulant, and antithrombotic effect that contributes to maintaining platelet homeostasis during acute inflammation. Antiplatelet treatment has been suggested as a novel strategy in sepsis, and we speculate that promoting efficient removal of activated and apoptotic platelets could further improve patient outcomes. Therefore, clearance of activated platelets earlier in the disease process could hasten recovery of homeostasis in circulation by eliminating catalytic platforms for the coagulation pathway, protecting blood cells from excessive activation, and restoring their normal function. Endothelium, at least in part, contributes to platelet disposal and may further improve the hypercoagulable status in inflammation. It is noteworthy that PS-mediated and lactadherin-strengthened platelet engulfment may modify coagulopathy, and thus provide a new modality for treatment of septic clotting disorders. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Platelet-mimetic strategies for modulating the wound environment and inflammatory responses.

Platelets closely interface with the immune system to fight pathogens, target wound sites, and regulate tissue repair. Natural platelet levels within the body can be depleted for a variety of reasons, including excessive bleeding following traumatic injury, or diseases such as cancer and bacterial or viral infections. Platelet transfusions are commonly used to improve platelet count and hemostatic function in these cases, but transfusions can be complicated by the contamination risks and short storage life of donated platelets. Lyophilized platelets that can be freeze-dried and stored for longer periods of time and synthetic platelet-mimetic technologies that can enhance or replace the functions of natural platelets, while minimizing adverse immune responses have been explored as alternatives to transfusion. Synthetic platelets typically comprise nanoparticles surface-decorated with peptides or ligands to recreate specific biological characteristics of platelets, including targeting of wound and disease sites and facilitating platelet aggregation. Recent efforts in synthetic platelet design have additionally focused on matching platelet shape and mechanics to recreate the marginalization and clot contraction capabilities of natural platelets. The ability to specifically tune the properties of synthetic platelet-mimetic materials has shown utility in a variety of applications including hemostasis, drug delivery, and targeted delivery of cancer therapeutics.

Endothelial microparticles (EMP) in physiology and pathology

Endothelial microparticles (EMP) are released from endothelial cells (ECs) in the process of activation and/or apoptosis. They harbor adhesive molecules, enzymes, receptors and cytoplasmic structures and express a wide range of various constitutive antigens, typical for ECs, at their surface. Under physiological conditions the concentration of EMP in the blood is clinically insignificant. However, it was reported that under pathological conditions EMP concentration in the blood might slightly increase and contribute to blood coagulation, angiogenesis and inflammation. It has been shown that EMP directly and indirectly contribute to the activation of blood coagulation. Endothelial microparticles directly participate in blood coagulation through their surface tissue factor (TF) - a major initiator of blood coagulation. Furthermore, EMP exhibit procoagulant potential via expression of negatively charged phospholipids at their surface, which may promote assembly of coagulation enzymes (TF/VII, tenases and prothrombinase complexes), leading to thrombus formation. In addition, they provide a binding surface for coagulation factors: IXa, VIII, Va and IIa. Moreover, it is possible that EMP transfer TF from TF-bearing EMP to activated platelets and monocytes by binding them through adhesion molecules. Also, EMP express von Willebrand factor, which may facilitate platelet aggregation. Apart from their procoagulant properties, it was demonstrated that EMP may express adhesive molecules and metalloproteinases (MMP-2, MMP-9) at their surface and release growth factors, which may contribute to angiogenesis. Additionally, surface presence of C3 and C4 - components of the classical pathway - suggests pro-inflammatory properties of these structures. This article contains a summary of available data on the biology and pathophysiology of endothelial microparticles and their potential role in blood coagulation, angiogenesis and inflammation.

Two disparate ligand-binding sites in the human P2Y1 receptor.

In response to adenosine 5'-diphosphate, the P2Y1 receptor (P2Y1R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y1R in complex with a nucleotide antagonist MRS2500 at 2.7 Å resolution, and with a non-nucleotide antagonist BPTU at 2.2 Å resolution. The structures reveal two distinct ligand-binding sites, providing atomic details of P2Y1R's unique ligand-binding modes. MRS2500 recognizes a binding site within the seven transmembrane bundle of P2Y1R, which is different in shape and location from the nucleotide binding site in the previously determined structure of P2Y12R, representative of another P2YR subfamily. BPTU binds to an allosteric pocket on the external receptor interface with the lipid bilayer, making it the first structurally characterized selective G-protein-coupled receptor (GPCR) ligand located entirely outside of the helical bundle. These high-resolution insights into P2Y1R should enable discovery of new orthosteric and allosteric antithrombotic drugs with reduced adverse effects.

CFTR regulates acute inflammatory responses in macrophages.

Mutation of cystic fibrosis transmembrane conductance regulator (CFTR) in the airway epithelial cells can lead to recurrent airway inflammation in cystic fibrosis (CF). Dysfunction of CFTR in neutrophils could contribute to LPS-induced acute lung inflammation. Deficiency of CFTR could also facilitate platelet aggregation and neutrophil-platelet interaction and promote inflammation. To study whether inhibition or mutation of CFTR in alveolar macrophages (AMs) or peritoneal macrophages (PMs) would promote their proinflammatory responses and whether dysfunction of CFTR would deteriorate acute E. coli-induced lung or peritoneal inflammation. Laboratory study. ELISA was used to determine production of proinflammatory cytokines in the CFTR inhibited or mutated macrophages under LPS challenge. Lung or peritoneum lavage was used to analyze proinflammatory parameters and cell differentiation. Excess lung water and lung vascular permeability were measured for evaluating severity of acute lung inflammation. Escherichia coli LPS simulation in AMs increased CFTR expression. Inhibition or mutation of CFTR in both AMs and PMs enhanced production of tumor necrosis factor alpha (TNF-α) and macrophage inflammatory protein-2 (MIP-2). Mutation of CFTR in macrophages exaggerated production of cytokines through NF-kB and p38 MAPK. Inhibition of CFTR by MalH2 or CFTRinh-172 deteriorates E. coli-induced acute lung inflammation. Deficiency of CFTR promotes migration of monocytes and neutrophils in E. coli pneumonia and peritonitis mouse models. CFTR expressed by alveolar or peritoneal macrophages regulates acute proinflammatory responses.

Group VIB calcium-independent phospholipase A2 (iPLA2γ) regulates platelet activation, hemostasis and thrombosis in mice.

In platelets, group IVA cytosolic phospholipase A2 (cPLA2α) has been implicated as a key regulator in the hydrolysis of platelet membrane phospholipids, leading to pro-thrombotic thromboxane A2 and anti-thrombotic 12-(S)-hydroxyeicosatetranoic acid production. However, studies using cPLA2α-deficient mice have indicated that other PLA2(s) may also be involved in the hydrolysis of platelet glycerophospholipids. In this study, we found that group VIB Ca2+-independent PLA2 (iPLA2γ)-deficient platelets showed decreases in adenosine diphosphate (ADP)-dependent aggregation and ADP- or collagen-dependent thromboxane A2 production. Electrospray ionization mass spectrometry analysis of platelet phospholipids revealed that fatty acyl compositions of ethanolamine plasmalogen and phosphatidylglycerol were altered in platelets from iPLA2γ-null mice. Furthermore, mice lacking iPLA2γ displayed prolonged bleeding times and were protected against pulmonary thromboembolism. These results suggest that iPLA2γ is an additional, long-sought-after PLA2 that hydrolyzes platelet membranes and facilitates platelet aggregation in response to ADP.

Soluble Trem-like Transcript-1 Regulates Leukocyte Activation and Controls Microbial Sepsis

The triggering receptor expressed on myeloid cells (TREM)-1 plays a crucial role during the onset of sepsis by amplifying the host immune response. The TREM-like transcript-1 (TLT-1) belongs to the TREM family, is selectively expressed on activated platelets, and is known to facilitate platelet aggregation through binding to fibrinogen. In this study, we show that a soluble form of TLT-1 is implicated in the regulation of inflammation during sepsis by dampening leukocyte activation and modulating platelet-neutrophil crosstalk. A 17-aa sequence of the TLT-1 extracellular domain (LR17) is responsible for this activity through competition with the TREM-1 ligand. Whereas early or late LR17 treatment of septic mice improves survival, treml-1(-/-) animals are highly susceptible to polymicrobial infection. The present findings identify platelet-derived soluble TLT-1 as a potent endogenous regulator of sepsis-associated inflammation and open new therapeutic perspectives. We anticipate soluble TLT-1 to be important in regulating leukocyte activation during other noninfectious inflammatory disorders.