Systems Biology of Platelet Activation

Platelet intracellular calcium mobilization [Ca(t)]i is a measure of platelet activation and controls important events downstream that contribute to hemostasis such as granule release, cyclooxygenase-1 and integrin activation, and phosphatidylserine exposure. Accurate simulations of blood clotting events require prediction of platelet [Ca(t)]i in response to combinatorial agonists. Therefore, a data-driven human platelet calcium calculator was developed using neural network (NN) ensemble trained on pairwise agonist scanning (PAS) data. PAS deployed all single and pairwise combinations of six important agonists (ADP, convulxin, thrombin, U46619, iloprost and GSNO used at 0.1, 1, and 10xEC50 to stimulate platelet P2Y1/P2Y12, GPVI, PAR1/PAR4, TP, IP receptors, and guanylate cyclase, respectively, in Factor Xa-inhibited (250 nM apixaban), diluted platelet rich plasma. PAS of 10 healthy donors (5 male, 5 female) provided [Ca(t)]i data for training 10 neural networks (NN, 2-layer/12-nodes) per donor. Trinary stimulations were then conducted at all 0.1x and 1xEC50 doses (160 conditions) as was a sampling of 45 higher ordered combinations (four to six agonists). The NN-ensemble average accurately predicted [Ca (t)]i beyond the single and binary training set for trinary stimulations (R = 0.924). The 160 trinary synergy scores, a normalized metric of signaling crosstalk, were also well predicted (R = 0.850) as were the calcium dynamics (R = 0.871) and high-dimensional synergy scores (R = 0.695) for the 45 higher ordered conditions. The calculator even predicted sequential addition experiments (n = 54 conditions, R = 0.921). NN-ensemble is a fast calcium calculator that proved to be useful for multiscale clotting simulations that include spatiotemporal concentrations of ADP, collagen, thrombin, thromboxane, prostacyclin, and nitric oxide. From sequential addition experiments done in PAS, it was discovered that activating platelets with thrombin in platelet-rich plasma (PRP) caused an attenuation of convulxin-induced, GPVI platelet receptor-mediated, calcium mobilization when convulxin was added to PRP approximately six minutes later. This attenuation effect was not observed when ADP and thromboxane analog, U46619 was used in place of thrombin. When PAR-1 and PAR-4 receptor agonists (AYPGKF and SFLLRN) were used instead of thrombin for the initial dispense, the subsequent convulxin-induced calcium response was also unaffected, demonstrating thrombin’s unique role in causing attenuation of subsequent convulxin-induced calcium mobilization. Thrombin, unlike ADP, U46619 or the PAR-1 and PAR-4 receptor agonists, is able to polymerize fibrinogen into fibrin. When GPRP was added to prevent polymerization of fibrin, initial platelet activation by thrombin did not result in attenuation of convulxin- induced calcium mobilization. This experiment was repeated using a mixture of washed platelets and fibrinogen monomers instead of PRP and yielded similar results. The presence of polymerized fibrin also reduced platelet deposition in a microfluidic assay on a collagen surface. These results suggest that polymerized fibrin binds to and downregulates platelet GPVI, a platelet receptor that is important to thrombus growth and is central to mediating hemostasis

Multiscale Modelling Of Platelet Aggregation

During clotting under flow, platelets bind and activate on collagen and release autocrinic factors such ADP and thromboxane, while tissue factor (TF) on the damaged wall leads to localized thrombin generation. Toward patient-specific simulation of thrombosis, a multiscale approach was developed to account for: platelet signaling (neural network trained by pairwise agonist scanning, PAS-NN), platelet positions (lattice kinetic Monte Carlo, LKMC), wall-generated thrombin and platelet-released ADP/thromboxane convection-diffusion (PDE), and flow over a growing clot (lattice Boltzmann). LKMC included shear-driven platelet aggregate restructuring. The PDEs for thrombin, ADP, and thromboxane were solved by finite element method using cell activation-driven adaptive triangular meshing. At all times, intracellular calcium was known for each platelet by PAS-NN in response to its unique exposure to local collagen, ADP, thromboxane, and thrombin. The model accurately predicted clot morphology and growth with time on collagen/TF surface as compared to microfluidic blood perfusion experiments. The model also predicted the complete occlusion of the blood channel under pressure relief settings. Prior to occlusion, intrathrombus concentrations reached 50 nM thrombin, ~1 μM thromboxane, and ~10 μM ADP, while the wall shear rate on the rough clot peaked at ~1000-2000 sec-1. Additionally, clotting on TF/collagen was accurately simulated for modulators of platelet cyclooxygenase-1, P2Y1, and IP-receptor. The model was then extended to a rectangular channel with symmetric Gaussian obstacles representative of a coronary artery with severe stenosis. The upgraded stenosis model was able to predict platelet deposition dynamics at the post-stenotic segment corresponding to development of artery thrombosis prior to severe myocardial infarction. The presence of stenosis conditions alters the hemodynamics of normal hemostasis, showing a different thrombus growth mechanism. The model was able to recreate the platelet aggregation process under the complex recirculating flow features and make reasonable prediction on the clot morphology with flow separation. The model also detected recirculating transport dynamics for diffusible species in response to vortex features, posing interesting questions on the interplay between biological signaling and prevailing hemodynamics. In future work, the model will be extended to clot growth with a patient cardio-vasculature under pulsatile flow conditions

Dual antiplatelet and anticoagulant (APAC) heparin proteoglycan mimetic with shear-dependent effects on platelet-collagen binding and thrombin generation

Heparin proteoglycans (HEP-PGs) carry standard heparin-mediated anticoagulant properties as well as novel antiplatelet functions, a combination that may be significant for targeting multiple pathways in a single therapy. Recent work developing semisynthetic HEP-PG mimetics has shown promising results also in vivo, however flow conditions in vitro that replicate in vivo hemodynamics have not been reported. In this work, we present several assays (platelet calcium mobilization, aggregometry, microfluidic tests at venous and arterial hemodynamics) to characterize specific mechanistic effects of dual antiplatelet and anticoagulant (APAC) constructs as mimetics of HEP-PGs. Three APACs with different conjugation levels of heparin chains (CL10, CL18, HICL) were shown to decrease platelet deposition to collagen surfaces in PPACK-treated whole blood at venous shear rate (200 s(-1)). FXIIa-inhibited whole blood (CTI: corn trypsin inhibitor, 40 mu g/mL) perfused over collagen/tissue factor showed reduced both platelet and fibrin deposition when treated with APACs. IC50 values for platelet and fibrin inhibition were calculated for each molecule at venous shear rate. Increasing the shear rate to arterial flows (1000 s(-1)) and using APAC as the sole anticoagulant, resulted in a more potent antiplatelet effect of APAC, suggesting an added effect on von Willebrand Factor (vWF) function. Additionally, APAC caused an inhibition of calcium mobilization specific to thrombin and collagen stimulation and a dose-dependent reduction in collagen-mediated platelet aggregation. Understanding the sensitivity of APAC activity to shear rate, platelet signaling and procoagulant pathways is important for applications in which APAC administration may have beneficial therapeutic effects.Peer reviewe

A Computational Approach To The Study Of Trauma

Trauma with hypovolemic shock is an extreme pathological state that challenges the body to maintain blood pressure and oxygenation in the face of hemorrhagic blood loss. In conjunction with surgical actions and transfusion therapy, survival requires the patient’s blood to maintain hemostasis to stop bleeding. The physics of the problem are multiscale: (1) the systemic circulation sets the global blood pressure in response to blood loss and resuscitation therapy, (2) local tissue perfusion is altered by localized vasoregulatory mechanisms and bleeding, and (3) altered blood and vessel biology resulting from the trauma as well as local hemodynamics control the assembly of clotting components at the site of injury. Building upon ongoing modeling efforts to simulate arterial or venous thrombosis in a diseased vasculature, we have developed models of trauma (both multiscale and machine-learning based) to understand patient risk and predict response. Key results were: (1) the upstream vascular network rapidly depressurizes to reduce blood loss, (2) wall shear rates at the hemorrhaging wound exit are sufficiently high (~10,000 s-1) to drive von Willebrand Factor unfolding, (3) full coagulopathy results in \u3e2L blood loss in 2 hours for severing all vessels of 0.13 to 0.005 mm diameter within the bifurcating network, whereas full hemostasis limits blood loss to \u3c100 mL within 2 min, and (4) hemodilution from transcapillary refill increases blood loss and could be implicated in trauma induced coagulopathy. Machine learning based methods were also implemented to understand trauma patient outcomes. A 400-estimator gradient boosting classifier was trained to predict survival probability and the model is able to predict a survival probability for any trauma patient and accurately distinguish between a deceased and survived patient in 92.4% of all cases. Partial dependence curves (Psurvival vs. feature value) obtained from the trained model revealed the global importance of Glasgow coma score, age, and systolic blood pressure while pulse rate, respiratory rate, temperature, oxygen saturation, and gender had more subtle single variable influences. Shapley values, which measure the relative contribution of each of the 8 features to individual patient risk, were computed for several patients and quantified patient-specific warning signs

Computational models of hemostasis: Degrees of complexity

The history of studies on blood clotting goes back to the emergence of civilized society itself. The foundations of the modern scientific study of hemostasis are based on the discovery of erythrocytes in blood in 1674 and, later, that of platelets in 1842. The causes of thrombosis are encapsulated in the Virchow Triad (dated to 1856), which refers, in modern terms, to hypercoagulability, alterations of hemodynamics (stasis), and endothelial injury. The understanding of coagulation, the network of reactions that underlies hemostasis and thrombosis, has evolved from a cascade (in 1964) into spatially distinct sets of reactions dependent on co-factors occurring on different cells in different tissues and linked together by diffusion and flow (as of 2015). Correspondingly, mathematical/computational models for hemostasis and thrombosis (which involve coagulation along with platelet aggregation in the presence of flow) have evolved in design complexity from Continuum temporal (or “homogeneous”) models to Continuum spatio-temporal models (with or without the flow) and lately into Discrete-Continuum spatio-temporal models with the flow. After a brief listing of the discoveries and historical personae that contributed to the understanding of hemostasis up to the present, the development of mathematical/computational models is traced from the late 1980s when they started gaining importance. Influential models are then highlighted. The models are reviewed in increasing order of design complexity (one of four possible methods of classification). The physiological significance of each and the insights they offer into hemostasis regulation are explained. © 2022 The Author

Effect of Endogenous and Exogenous Agents in Platelet Adhesion

Platelet adhesion is regulated by both activators, such as adenosine diphosphate, and inhibitors, such as nitric oxide (NO). Both agents are released on platelet activation, so that platelets initiate both positive and negative feedback systems. In vivo platelet adhesion models generally consider the effects of platelet activators and inhibitors separately. The goal of this study was to create an environment in which interplay between positive and negative feedback can be observed together and in which the roles of endogenous and exogenous platelet activators are distinguishable. The results are expected to be applicable to the design of stents, which are susceptible to thrombus formation, and which provide multiple adjacent regions where platelet-released agents can interact with one another. To distinguish between the role of exogenous and endogenous agents, microchannels were produced that had multiple thrombogenic (fibrinogen) regions separated by non-thrombogenic (BSA-coated) regions. This geometry reveals the effect of agents released from different thrombogenic regions on one another. Adhesion was quantified by percent platelet surface area coverage. Surface area coverage differed between the upstream and downstream sides of the thrombogenic regions. Positive and negative feedback effects were enhanced by increased platelet production of activator and inhibitor. In contrast, when the NO donor DPTA NONOate was added, with the intent to overwhelm the endogenous feedback, a more uniform spatial distribution of adhesion was obtained. Though these agonists, activator (ADP) and inhibitor (NO), act on different receptors through different signaling pathways, they all lead to increase or decrease in the intracellular Ca2+ concentration ([Ca2+]) depending on the agonist produced. Ca2+ is a key component and serves as second messenger in all cells regulatory processes. The increase in [Ca2+] leads to several steps of activation. The process leading Ca2+ to increase and decrease and its underlying mechanisms remained largely unknown.To examine Ca2+ response, platelets were loaded with the Fluo-4 Ca2+ ion indicators and fluorescence waveform was monitored. We found that Ca2+ response increased dose dependently with addition of an activator (ADP) and addition of endogenous (L-Arginine) and exogenous (DPTA NONOate) NO donor showed decrease in Ca2+ response in ADP stimulated platelets

The Effects Of Platelet Signaling Inhibitors On Clot Development Under Flow

GPVI is the first responder towards collagen surface and therefore, the role of GPVI in facilitating primary platelet deposition is well studied and unquestionable. However, whether it plays a part in secondary platelet deposition by binding with fibrin, thus facilitating further platelet activation remains a question. Depending on the experiment method and rationale behind it, different results and conclusion could be achieved, thus it calls for a method that could better recapitulate human blood system under in vitro setting with appropriate methods to inhibit GPVI.Indirect method incorporates Syk and Src family kinases (SFK) inhibitors; these molecules interfere with signaling from GPVI, α2β1, αIIbβ3, and GPIb-IX-V to reduce thrombotic risk or induce bleeding episodes. Collagen-mediated clustering of platelet GPVI results in phosphorylation of SFKs such as Lyn and Fyn, and active Lyn is constitutively bound to GPVI to allow rapid signaling. During clotting under flow, the generation of fibrin can have diverse influences on platelet signaling by sequestering thrombin and potentially activating GPVI signaling within the clot interior. These inhibitors tackle the thrombus formation at earlier stages since the platelets reach the activation surface. Direct inhibition of GPVI, which involves using an artificial anti-GPVI fragment, was used to avoid undesirable inhibition and compare the difference between inhibition of subsequent pathways. Using microfluidics, the effects of these inhibitors can be explored under defined hemodynamic flows and procoagulant surface triggers. Additionally, the drug may be present in the blood at desired time of clotting by perfusion switching to drug-treated blood. This experimental design allows exploration of platelet response at different stages of clotting through the measurement of drug potency to modulate clotting on different procoagulant surface conditions, interactions between various coagulation factors in plasma, and the kinetics of several competitive reactions to facilitate platelet recruitment, granule release and fibrin formation

Microfluidic Approaches to Thrombosis and Hemostasis: Towards a Patient-Specific Test of Antiplatelet Therapeutics and the Assessment of Coagulopathy in Hemophilic and Trauma Patients

Current in vitro or ex vivo models of hemostasis and thrombosis fail to recapitulate the hemodynamic conditions and biorheologic phenomena found throughout the vasculature. Microfluidic technology enables physiologic hemodynamics for the study of platelet deposition and coagulation using minimum volumes of human whole blood. This dissertation describes the application of microfluidic assays, the manipulation of surface-patterned procoagulant and sub-endothelial proteins, anti-coagulation, and flow conditions to investigate platelet function and coagulation under flow. First, we demonstrate a novel method to assess the in vivo or in vitro therapeutic efficacy of anti-platelet therapies on platelet aggregates adhering to collagen type I surfaces. We phenotyped individual healthy donor platelet function responses to in vivo or in vitro aspirin, a common antiplatelet therapy over collagen type I surfaces at venous shear rates. Utilizing the same flow assay, we also characterized mechanism-based resistance to aspirin conferred by non-steroidal anti-inflammatory drugs. Furthermore, we have also developed a new model to assess the intrinsic pathway of coagulation under flow on collagen type I surfaces and investigated the role of the intrinsic pathway in recombinant coagulation factor VIIa (rFVIIa) therapeutic efficacy. We then extended this mechanistic investigation of rFVIIa to flow assays where clotting is initiated by collagen and immobilized lipidated tissue factor to evaluate the role of the intrinsic tenase in conjunction with exogenous rFVIIa when surface-triggered extrinsic pathway is present. Finally, we continued to assess coagulopathic patients by first mimicking resuscitation-driven hemodilution, hyperfibrinolysis, and plasmin-inhibitor therapy under flow. We then evaluated downregulation of platelet function in whole blood from trauma patients during the acute phase of trauma-induced coagulopathy. The development of microfluidics, microfabrication, and its applications in hemostasis and thrombosis is essential in advancing our knowledge of clinical and pathological disorders such as myocardial infracts, hemophilia, and deep vein thrombosis. Beyond this work, microfluidic platforms in hemostasis and thrombosis can potentially be used as drug screening platforms for antiplatelet or clotting factor therapies, or a point of care diagnostic test for bleeding and pin-pointing the therapeutic index of novel biopharmaceutics

Targeted Imaging Agents for Detecting Tumour Cell Death following Therapy

Cell death is an important target for imaging the early response of tumours to treatment. In this study a phosphatidylserine-binding protein (C2Am) has been derivatised with a fluorine-18 containing maleimide for imaging tumour cell death in a xenograft murine advanced colorectal cancer. A one-pot, two-step automated synthesis of N-(5-[18F]fluoropentyl)maleimide using a GE TRACERlab FXFN automated module (within 58±5.8 min (n = 12), >98% radiochemical purity and 12±3% decay corrected yield) has been developed. This was used to label the single cysteine present on C2Am within 30 min in PBS (Am=212000±30000 MBq/µmol (n = 3)). Using xenograft models of breast and colorectal cancer, and a TRAIL-R2 agonist for inducing cell death, the binding of [18F]FPenM-C2Am was tested in vitro and in vivo using biodistribution and dynamic PET imaging studies. Cell death detection was validated by cell death histology assays CC3 and TUNEL. For colorectal cancer, there was a positive correlation between [18F]FPenM-C2Am signal in tumours post treatment and the corresponding histologic markers of cell death CC3 (Pearson r = 0.82) and TUNEL (Pearson r = 0.95). [18F]FPenM-C2Am showed a favourable biodistribution profile, with predominantly renal clearance and minimal retention in spleen (0.79±0.05 %ID/g), liver (1.18±0.13 %ID/g), small intestine (0.97±0.25 %ID/g) and kidney (6.90±0.56 %ID/g) at 2 h after probe administration. In a xenograft model of colorectal cancer treated with a TRAIL-R2 agonist, for 24h, at 0.2-0.4 mg/kg, i.v. [18F]FPenM-C2Am generated tumour-to-muscle and tumour-to-blood ratios following treatment of 6.7±0.8-fold and 1.89±0.23-fold, respectively, at 2 h after administration. A statistically significant pairwise difference was obtained between the tumour-to-muscle contrast prior to and following therapy (P=0.0137, unpaired two-tailed t-test). Given the favourable biodistribution profile of [18F]FPenM-C2Am, and its ability to produce rapid and cell death-specific image contrast, this agent has potential for clinical translation. We have initiated cGMP manufacture and toxicology studies required for a Phase 1 trial. Aberrant cell surface glycosylation has been described as one of the key hallmarks of cancer. Monitoring glycosylation could provide an insight into tumour progression, proliferation and ultimately could potentially be used for monitoring treatment response. Aberrant glycosylation can be observed by harnessing the cell’s metabolism to incorporate into its glycome unnatural sugars bearing bioorthogonal chemical reporters. These reporters are targeted subsequently by fluorescent, magnetic or optoacoustic probes that allow imaging. As part of this work, we have demonstrated in vitro that a peracetylated cyclopropene mannosamine (Ac4ManNCCp)-modified sugar can be used as a tool for imaging hypersialylation in an advanced colorectal cancer model. Further optimisation of the probe to improve solubility is required to facilitate transitioning from in vitro to in vivo imaging. Nevertheless, overcoming this solubility issue would allow for facile labelling of surface glycans using PET radionuclides

Ethnic differences in endothelial function and monocyte subsets in heart failure

Introduction and Aims: The progressive nature of heart failure (HF) is reflected by its complex pathophysiology, featured by imbalance of damaging and reparative factors. The overall aim was to assess the implication of endothelial (dys)function, monocyte subsets, different types of endothelial progenitors and plasma microparticles in subjects with HF. A special focus was an investigation of possible ethnic differences in these parameters. Methods: Parameters of vascular function, monocyte subsets, endothelial progenitors, and cellular microparticles were compared between South Asian subjects with systolic HF, and those with heart disease without HF and healthy controls. Ethnic differences in HF were assessed in three ethnic groups: South Asians, Whites, and African-Caribbeans. Additionally, leukocyte counts were compared between subjects with HF with reduced or preserved ejection fraction, whose outcome (mortality) was recorded during follow-up. Results: South Asian subjects with HF had significantly impaired micro- and macrovascular endothelial function, reduced levels of endothelial progenitors, and monocytes with reparative potential, but increased levels of microparticles. In HF patients, a high count of monocyte microparticles was associated with low ejection fraction. There were significant ethnic differences in characteristics of microvascular endothelial function, counts of CD14++CD16+ and CD14+CD16++ monocytes and monocyte-derived endothelial progenitors. On multivariate analysis, a high monocyte count was a significant predictor of death in HF with preserved ejection fraction unlike in those with systolic HF. Conclusions: Significant impairment of microvascular endothelial function is present in South Asian subjects with HF. High monocyte count is an independent predictor of death in HF with preserved ejection fraction. The value of the tested biological markers as therapeutic targets should be explored in future studies