Understanding how a highly prevalent <i>GRK5</i> polymorphism affects platelets and enhances thrombotic risk

Blood · 2026-01-20 · 4 citations

ABSTRACT: Inherited genetic variants that modulate platelet function contribute significantly to thrombotic disorders, yet their mechanisms and clinical implications remain underexplored. Two genome-wide association studies identified an A→G variant (rs10886430) in the first intron of G protein-coupled receptor kinase 5 (GRK5), found in homozygosity in ∼5 million Americans. The homozygous GRK5 GG genotype is associated with an increased risk of stroke and venous thromboembolism, but the mechanistic link between this variant and thrombotic risk has remained unclear. To investigate this, we identified 3 GG individuals. GRK5 protein levels in GG platelets were 90% lower than in AA controls. The significant reduction in GRK5 levels in GG platelets led to elevated platelet responsiveness to thrombin and a protease-activated receptor 1 (PAR1) agonist but not a PAR4 agonist. These findings were corroborated in GRK5-/- induced pluripotent stem cell-derived megakaryocytes, transgenic Grk5-deficient murine platelets, and AA platelets exposed to a GRK5 inhibitor. We demonstrated that PAR1 internalization was reduced in GG platelets, leading to enhanced PAR1 signaling. Under venous shear in an endothelialized microfluidic system, GG platelets exhibited increased accumulation, which was reversed by PAR1 inhibition with vorapaxar. In an arterial murine thrombosis model following human platelet infusion, GG platelets also showed enhanced thrombus formation in vivo. This study provides, to our knowledge, the first experimental evidence directly linking a highly prevalent human GRK5 variant to defective PAR1 regulation and increased thrombotic risk. Together, these findings establish that the GRK5 GG genotype confers increased thrombotic potential through impaired PAR1 desensitization, providing mechanistic insight that connects human genetics, thrombin receptor signaling, and thrombotic disease.

von Willebrand Factor fibers formed at pathological high shear provide a scaffold for α-synuclein binding and aggregation

Scientific Reports · 2026-04-25

Rapid Determination of Xa Inhibitor Activity in Blood Using a Microfluidic Device that Measures Platelet Deposition and Fibrin Generation Under Flow

TH Open · 2025-03-25

Abstract Patients taking direct oral anticoagulants (DOACs) often present complicated scenarios following major bleeding, stroke, or emergency surgery. Rapid whole blood assays of DOAC levels would aid clinical decisions such as the need for DOAC reversal. We developed a single-use, storage-stable, eight-channel microfluidic device to estimate factor Xa (FXa) inhibitor (apixaban or rivaroxaban) levels in venous thromboembolism or atrial fibrillation patients. The assay simultaneously measured whole blood clotting dynamics on collagen/tissue factor (TF; wall shear rate, 200−1) under four ex vivo conditions: no-treatment control, high dose Factor Xa inhibition, low dose or high dose FXa reversal agent (andexanet alfa). Fibrin and platelet deposition dynamics were monitored via two-color epifluorescence microscopy. Plasma samples were also evaluated by LC-MS/MS for DOAC concentrations. Experiments with healthy volunteer blood spiked with DOAC verified device performance (DOAC IC50 ∼120 nM) and confirmed that andexanet alfa added to healthy donor blood had no off-target effect on platelet or fibrin signal. Patient whole blood monitored for 15 to 25 minutes (17 minutes mean runtime) allowed calculation of functional DOAC concentrations ranging from 2 to 500 nM that correlated well with LC-MS/MS determination of apixaban or rivaroxaban (R2 = 0.7 or 0.9, respectively). Platelet dysfunction was not observed in any patient on DOAC. For a threshold of 100 nM DOAC, the area under the curve (AUC) was found to be 0.881 for apixaban and 0.933 for rivaroxaban. Microfluidic testing of whole blood can provide a rapid estimate of DOAC levels over the on-therapy range.

S4282 Recurrent Squamous Cell Carcinoma Presenting as Refractory Esophageal Stricture

The American Journal of Gastroenterology · 2025-10-01

Introduction: Esophageal strictures are a recognized complication of surgery and chemoradiation for head and neck malignancies. Although typically benign, resistance to endoscopic dilation should raise suspicion for malignancy. Recurrent or primary squamous cell carcinoma (SCC) may present subtly and elude detection on initial endoscopic biopsy. This case highlights the importance of repeated histologic evaluation in refractory strictures. Case Description/Methods: A 79-year-old man with Stage III (T2N1) supraglottic laryngeal SCC was treated with definitive chemoradiation, including cisplatin, in early 2020. Post-treatment positron emission tomography/computed tomography showed resolution of the primary tumor and regional nodes. He remained stable with persistent hoarseness until July 2024, when progressive dysphagia to solids began. Initial esophagogastroduodenoscopy (EGD) revealed a benign-appearing stricture 21–30 cm from the incisors, with a luminal diameter of 7 mm. Through the scope balloon dilation was performed to 12 mm, with mucosal disruption observed. Biopsy showed reactive squamous epithelium without malignancy or eosinophilia. Four serial EGDs over 8 weeks with through the scope balloon dilations up to 12 mm provided minimal improvement. The diameter remained at 8 mm on his fifth EGD prompting repeat biopsies showing moderately differentiated SCC. The patient opted for palliative management. Discussion: This case highlights the difficulty of distinguishing benign radiation-induced esophageal strictures from recurrent malignancy. Refractory strictures—defined as failure to reach or maintain a luminal diameter ≥14 mm after 5 dilation sessions spaced 2 weeks apart—warrant further investigation. Sampling error is common and diagnostic yield increases significantly when at least 6 samples are taken from 4 quadrants of the stricture. Advanced imaging modalities can improve diagnostic accuracy. Endoscopic ultrasound offers better delineation of mural and periesophageal involvement than computed tomography or positron emission tomography. Newer techniques such as autoantibody panels and volumetric laser endomicroscopy-guided biopsy may enable earlier detection of recurrent SCC in patients. In any patient with esophageal strictures refractory to dilation or a failure to improve >3 mm after 2 EGDs, especially those with prior head and neck cancer, malignancy should be suspected and repeat biopsies should be obtained. Early repeated biopsy is critical for timely diagnosis and management, particularly in high-risk individuals with prior chemoradiation exposure.

From imaging to computational domains for physics-driven molecular biology simulations: Hindered diffusion in platelet masses

bioRxiv (Cold Spring Harbor Laboratory) · 2025-02-08

Abstract When formed in vivo, murine hemostatic thrombi exhibit a heterogeneous architecture comprised of distinct regions of densely and sparsely packed platelets. In this study, we utilize high-resolution electron microscopy alongside machine learning and physics-based simulations to investigate how such clot microstructure impacts molecular diffusivity. We used Serial Block Face – Scanning Electron Microscopy (SBF-SEM) to image select volumes of hemostatic masses formed in a mouse jugular vein, producing large stacks of high-resolution 2D images. Images were segmented using machine learning software (Cellpose), whose training was augmented by manually segmented images. The segmented images were then utilized as a computational domain for Lattice Kinetic Monte-Carlo (LKMC) simulations. This process constitutes a computational pipeline that combines purely data-derived biological domains with physics-driven simulations to estimate how molecular movement is hindered in a hemostatic platelet mass. Using our pipeline, we estimated that the hindered diffusion rates of a globular protein range from 2% to 40% of the unhindered rate, with denser packing regions lending to lower molecular diffusivity. These data suggest that coagulation reactions rates, thrombin generation and activity, as well as platelet releasate activity may be drastically impacted by the internal geometry of a hemostatic thrombus. Author Summary Hemostasis and coagulation are two exquisitely complex, intertwined, and tightly regulated biological processes. Dysregulation of either process may lead to severe health consequences or death. Coagulation reactions have been extensively studied under static laboratory conditions, which are different from in vivo conditions. It is therefore imperative to understand if and how the chemical reactions underlying coagulation are regulated by the environment where they occur. In vivo experimentation enables us to image hemostasis, but not chemical reactions. Physics-driven molecular simulations of chemical reactions can bridge the gap, provided the physical environment is correctly represented computationally. The present work serves as a much-needed foundation for image-to-computation for physics based molecular simulations in biological domains.

From imaging to computational domains for physics-driven molecular biology simulations: Hindered diffusion in platelet masses

PLoS Computational Biology · 2025-07-07

When formed in vivo, murine hemostatic thrombi exhibit a heterogeneous architecture comprised of distinct regions of densely and sparsely packed platelets. In this study, we utilize high-resolution electron microscopy alongside machine learning and physics-based simulations to investigate how such clot microstructure impacts molecular diffusivity. We used Serial Block Face - Scanning Electron Microscopy (SBF-SEM) to image select volumes of hemostatic masses formed in a mouse jugular vein, producing high-resolution 2D images. Images were segmented using machine learning software (Cellpose), whose training was augmented by manually segmented images. The segmented images were then utilized as 2D computational domains for Lattice Kinetic Monte-Carlo (LKMC) simulations. This process constitutes a computational pipeline that combines purely data-derived biological domains with physics-driven simulations to estimate how molecular movement is hindered in a hemostatic platelet mass. Using our pipeline, we estimated that the 2D hindered diffusion rates of a globular protein range from 2% to 40% of the unhindered rate, with denser packing regions lending to lower molecular diffusivity. These data suggest that coagulation reactions rates, thrombin generation and activity, as well as platelet releasate activity may be drastically impacted by the internal geometry of a hemostatic thrombus.

S3378 Iatrogenic Pancreatic Duct Injury Following Left Nephrectomy

The American Journal of Gastroenterology · 2025-10-01

Introduction: Iatrogenic pancreatic duct injury is a rare but serious complication of left nephrectomy, particularly in the setting of dense retroperitoneal adhesions. Disruption of the pancreatic duct may lead to significant morbidity including external pancreatic fistula, pseudocyst formation and portosplenic venous thrombosis. Case Description/Methods: Fifty-three-year-old man with ankylosing spondylitis and recurrent left ureteropelvic junction obstruction complicated by recurrent pyelonephritis, underwent open left nephrectomy. Intraoperatively, dense adhesions and scar tissue involving the spleen and pancreatic tail were encountered due to patient's history of recurrent infections. Three months postoperatively, the patient developed a large retroperitoneal fluid collection (13 × 13 × 16 cm), which was initially managed with percutaneous drainage. However, following eventual removal of the drains approximately 5 months post-op, recurrent collections ensued requiring JP drain to be replaced. Further workup including fluid analysis revealed markedly elevated amylase (>7500 U/L) and lipase (>3000 U/L), consistent with pancreatic leak. Magnetic resonance cholangiopancreatography (MRCP) done at the time demonstrated a thick-walled collection near the pancreatic tail without overt ductal dilation or parenchymal injury, and resolution of fluid collection was noted after drain replacement on subsequent imaging. At 9 months post-op, patient had a followup computed tomography abdomen pelvis with contrast which revealed an isolated remnant of the distal pancreatic tail with a disconnected pancreatic duct, consistent with Disconnected Pancreatic Duct Syndrome (DPDS), along with a persistent pseudocyst (4.8 × 2.7 × 5 cm) and chronic splenic vein thrombosis. Development of collateral circulation around the splenic hilum and gastric wall was noted, indicating evolving sinistral (left-sided) portal hypertension with isolated gastric varices. Discussion: This case highlights a rare but significant complication of left nephrectomy: iatrogenic pancreatic duct injury leading to DPDS. Early recognition and multidisciplinary management involving gastroenterology, interventional radiology, and surgery are essential to prevent long-term morbidity. Pancreatic duct injury should be considered in patients with persistent high-amylase fluid collections following left-sided retroperitoneal surgery. DPDS is a challenging postoperative complication with potential for serious vascular and gastrointestinal sequelae.

S4101 Navigating Uncertainty: A Case of Colonic Polyposis and a PMS2 Variant of Uncertain Significance in Suspected Lynch Syndrome

The American Journal of Gastroenterology · 2025-10-01

Introduction: Lynch syndrome is an autosomal dominant inherited germline mutation in DNA mismatch repair (MMR) genes, including MLH1, MSH2, MSH6, and PMS2. It increases risk significantly for colon cancer and other malignancies. In patients presenting with early-onset of malignancy, ≥10 cumulative adenomatous polyps, or a family history fulfilling Amsterdam II criteria, identification of pathogenic MMR variants is essential for guiding screening and management of both patients and at-risk relatives. Genetic testing may reveal mutation variants of these genes with uncertain significance (VUS) complicating clinical decision-making. Case Description/Methods: A 77-year-old man with no known family history of colorectal cancer underwent a screening colonoscopy in 2020 showing a 2.5 cm tubulovillous adenoma requiring piecemeal resection and a 2 mm tubular adenoma. A colonoscopy in 2021 showed a 4 mm tubulovillous adenoma and 2 tubular adenomas less than 1 cm. Colonoscopy in 2024 showed a 4 cm tubular adenoma requiring piecemeal EMR and 7 additional tubular adenomas less than 1 cm. His polyp history prompted genetic testing. This revealed a mutation on chromosome 7 involving a PMS2 c.*3G >A VUS located in the 3′ untranslated region. This mutation is listed in ClinVar in association with Lynch syndrome, however it remains uncharacterized in the literature. Discussion: This case highlights the management challenges posed by a VUS in a gene classically associated with Lynch syndrome. VUS findings, such as PMS2 c.*3G >A, lack sufficient evidence for definitive risk stratification for colon cancer as opposed to pathogenic MMR variants which enable tailored surveillance. This specific non-coding variant may affect post-transcriptional regulation which would explain this patient’s polyp burden, however oncogenic potential remains unclear. Most PMS2-associated Lynch syndrome cases follow autosomal dominant inheritance, however biallelic mutations may result in constitutional mismatch repair deficiency (CMMRD), a rare autosomal recessive disorder. Alternatively, some non-inherited cases may result from 2 somatic mutations, known as double somatic hits. Surveillance decisions in patients with VUS mutations should be based on clinical history. Given the accelerated adenoma-to-carcinoma sequence associated with PMS2 mutations (1–2 years vs 7–10 years in sporadic cases), annual colonoscopic surveillance was recommended for this patient. Family members should also be informed and offered genetic counseling.

S3899 Fistulizing Crohn's Disease Causing Hydroureteronephrosis From External Compression by an Ileocolonic Fistula

The American Journal of Gastroenterology · 2025-10-01

Introduction: Fistula formation occurs in 20%-50% of patients with Crohn’s disease (CD). These most commonly occur as external fistulas (perianal or enterocutaneous) whereas internal fistulas account for only one third of cases. We present a case of an ileocolonic fistula causing acute kidney injury (AKI) from external compression of a ureter. Case Description/Methods: A 38-year-old-man with fistulizing CD, diagnosed in 2014 and previously treated with mesalamine monotherapy, presented with worsening abdominal pain. Computed tomography enterography revealed a fistula between the distal ileum and sigmoid colon, along with a fistula involving the distal ileum alone. There was moderate to severe right hydroureteronephrosis adjacent to inflammatory changes from the fistulas. There was mild wall thickening of the terminal ileum (TI). The findings are suggestive of external compression of the ureter from the ileocolonic fistulas. The patient was managed with right ureteral stent placement and initiated on steroids with subsequent improvement in renal function. Outpatient follow up with initiation of anti-TNF therapy was recommended. Discussion: AKI from a ureteral obstruction is a serious sequelae of inflammatory bowel disease. In CD, this can occur from active inflammation of bowel adjacent to the ureters, fistula or abscess formation causing external compression on the ureter, or from stones within the ureter. TI inflammation in CD impairs calcium absorption leading to hyperoxaluria, a risk factor for nephro/ureteronlithiasis. Treatment of the obstruction involves surgical placement of ureteral stents, or resection of the fistula or affected bowel in addition to medical management. In fistulizing CD, advanced therapies with anti-TNF agents and immunomodulators are typically used. Anti-interleukin antibody agents may also be an option in non-responders. There is little/no role in mesalamine for treatment in CD involving more than the left colon and this can precipitate AKI from acute interstitial nephritis or minimal change disease. Imaging should be pursued in all CD patients with AKI to evaluate for ureteral obstruction. Prompt diagnosis and management are essential to prevent irreversible renal damage.

Multiscale simulations that incorporate patient-specific neural network models of platelet calcium signaling predict diverse thrombotic outcomes under flow

PLoS Computational Biology · 2025-05-06 · 1 citations

During thrombosis, platelets rapidly deposit and activate on the vessel wall, driving conditions such as myocardial infarction and stroke. The complexity of thrombus formation in pathological flow geometries, along with patient-specific pharmacological responses, presents an opportunity for computational modeling to help deliver novel diagnostic and therapeutic insights. In the present study, we employed a multiscale 3D computational model that incorporates unique donor-derived neural networks (NNs) trained with platelet calcium mobilization traces under combinatorial exposure to 6 agonists (n = 10 donors). The 3D model comprises four modules: a donor-specific NN model for platelet calcium mobilization, a lattice kinetic Monte Carlo solver for tracking platelet motion and bonding, a finite volume method solver for modeling soluble agonist release and convective-diffusive transport, and a lattice Boltzmann method solver for predicting the blood velocity field. Simulations were conducted for platelets from individual blood donors under venous and arterial flow conditions on a defined collagen surface, examining the effects of inhibiting ADP and TXA2, as well as the influence of nitric oxide and prostacyclin. The results reveal significant individual variability in platelet responses, influencing simulated thrombus growth dynamics and emphasizing the importance of personalized models for predicting thrombotic behavior. This approach enables consideration of patient-specific platelet signaling, drug responses, and vascular geometry for predicting thrombotic episodes, essential for advancing precision medicine and improving patient outcomes in thrombotic conditions.