Anisocytosis, indicated by an elevated red blood cell distribution width (RDW), has been associated with poor cardiovascular outcomes. However, its relevance to thrombus composition and long-term outcomes in stroke patients undergoing thrombectomy remains unclear. We investigated whether anisocytosis is associated with histologic thrombus characteristics and long-term prognosis in this population.This retrospective multicenter cohort study included 401 patients with acute ischemic stroke who underwent intra-arterial thrombectomy between September 2014 and December 2020. Thrombus composition was assessed using immunohistochemistry to quantify the proportions of fibrin, red blood cells, platelets, and thrombin. Patients were followed for major adverse cardiovascular events, including all-cause mortality, ischemic heart disease, and recurrent ischemic stroke.Among the 401 patients (mean age, 73.1 years; 50.1% male), anisocytosis (RDW > 15.7%) was present in 67 (16.7%). Thrombi from patients with anisocytosis had higher platelet content (12.2% vs. 9.1%; P = 0.008) and lower erythrocyte content (30.0% vs. 37.3%; P = 0.007) than those without anisocytosis. During a median follow-up of 38.5 months, anisocytosis was independently associated with increased risk of recurrent ischemic stroke (hazard ratio, 2.61; 95% confidence interval, 1.12-6.10; P = 0.027), but not with overall major adverse cardiovascular events, mortality, or ischemic heart disease.Anisocytosis was associated with platelet-rich thrombi and an increased risk of stroke recurrence after thrombectomy. These findings suggest a possible role of red blood cells in platelet-mediated thrombosis and indicate that RDW may have potential prognostic value for identifying patients at higher risk for recurrent events.

Ischemic stroke requires effective reperfusion therapies to limit brain injury, yet rtPA (recombinant tissue-type plasminogen activator) efficacy is limited, particularly in platelet-rich thrombi. Neutrophil extracellular traps (NETs) and their components, especially histones and DNA, contribute to thrombolysis resistance. Chondroitin sulfate (CS), a glycosaminoglycan with high affinity for extracellular histones, may neutralize their prothrombotic effects and improve outcomes. This study aimed to evaluate the effects of CS in preclinical ischemic stroke models and its impact on components of neutrophil extracellular traps. Two mouse models of middle cerebral artery occlusion were used: a fibrin-rich thromboembolic stroke model (rtPA-sensitive) and a platelet-rich aluminum chloride model (rtPA-resistant). Mice received intravenous CS (30-120 mg/kg), rtPA (10 mg/kg), or a combination of both. Lesion volume, tissue recanalization/reperfusion, hemorrhagic transformation, and functional connectivity were assessed via 7T magnetic resonance imaging and ultrafast Doppler imaging. In vitro coagulation-fibrinolysis assays examined the effects of neutrophil extracellular trap components on fibrin polymerization and fibrinolysis, and their modulation by CS±rtPA. In the fibrin-rich model, CS alone reduced lesion volume by 36% and improved recanalization, comparable to rtPA (43%), without increasing hemorrhagic transformation. CS enhanced functional connectivity recovery at 24 hours, whereas combined CS+rtPA lost these benefits. In the platelet-rich model, CS did not affect lesion size, recanalization, or hemorrhage. In vitro, histones promoted clot stabilization and altered fibrinolysis, effects fully neutralized by equimolar CS in the absence of rtPA. With rtPA, CS's neutralizing capacity was reduced, and histone-driven profibrinolysis was accentuated at higher CS doses. DNA produced opposite effects to histones, and combined DNA+histones masked histone activity, resisting inhibition by CS+DNase. CS mitigates histone-mediated prothrombotic effects, improves reperfusion and network recovery in fibrin-rich stroke, but loses efficacy in platelet-rich thrombi and when combined with rtPA. These findings support CS as a potential adjunct or alternative therapy, particularly for patients with contraindications to rtPA.

Thrombolytic agents are serine proteases or related enzymes that facilitate blood clot breakdown via a process called thrombolysis. They work either indirectly, via activation of plasminogen into plasmin-a potent fibrin-degrading enzyme-or by direct dissolution of fibrin within clots. Numerous clinical trials investigating thrombolytic candidates for major thrombotic conditions, such as heart attack and acute ischaemic stroke (AIS), have led to the development of several potential therapies. However, while some drugs show clinical benefits, most still possess substantial limitations, especially for the treatment of AIS. Recombinant tissue-type plasminogen activator (rt-PA; alteplase) was the only clot-busting medication officially approved by the Food and Drug Administration for AIS from 1996 until 2024, until the recent approval of its mutated version, tenecteplase (2025). However, the use of rt-PA is significantly limited by its short plasma half-life, and both rt-PA and tenecteplase possess reduced efficacy against platelet-rich thrombi and a narrow, guideline-recommended therapeutic window up to 4.5 hours from stroke onset, primarily due to a diminished risk-benefit profile beyond this time point. While recent thrombectomy techniques offer ground-breaking ways for the removal of large clots, a pressing need remains to improve pharmacological thrombolysis. Novel strategies, including the fusion of clot-busting enzymes with thrombus-targeting antibodies and nanotechnology-based delivery systems, are being tested for their ability to increase the precision and safety of thrombolytic therapy. This review will articulate the historical milestones in thrombolysis, discuss key thrombolytic agents and their generational derivatives, and explore innovative approaches to advance this life-saving therapy for AIS.

Novel von willebrand factor targeting thrombolytic TGD001 is effective in diverse thrombotic models, demonstrating its potential as a universal thrombolytic

Novel von willebrand factor targeting thrombolytic TGD001 is safe and well tolerated in a phase 1 first-in-human trial

Von Willebrand factor (VWF) plays an important role in primary hemostasis. Dysregulated plasma VWF levels are implicated in various pathological conditions. Reduced or dysfunctional VWF is associated with bleeding, known as von Willebrand disease. Whereas elevated plasma VWF levels may give rise to an increased risk of developing arterial thrombotic events. In general, antithrombotic strategies in arterial thrombosis primarily focus on inhibiting platelet aggregation; however, treatment failure, antiplatelet drug resistance, and adverse bleeding tendencies underscore the necessity for the development of more efficacious and safer therapeutic modalities. Targeting VWF presents an interesting therapeutic approach as it operates independently of platelet activation pathways for platelet-rich thrombus formation. Over time, several VWF inhibitors have progressed to clinical application for thrombosis management, with ongoing research endeavors exploring novel compounds targeting VWF. This review provides a comprehensive overview of the evolution of VWF-targeting therapeutic agents, elucidating their current developmental stages, clinical indications, and evaluating their respective advantages and limitations.

r-tPA (recombinant tissue-type plasminogen activator) and its variant, TNK (tenecteplase), are the currently approved thrombolytic drugs for the treatment of acute ischemic stroke, but they are ineffective in a proportion of patients due to r-tPA resistance of platelet-rich thrombi. A novel thrombolytic, caADAMTS13 (constitutively active a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) has been shown to improve experimental stroke outcomes where platelet-rich thrombi are present but have not been directly compared with r-tPA or TNK. We conducted a direct comparison of caADAMTS13 versus r-tPA versus TNK versus vehicle control in the ferric chloride-mediated distal middle cerebral artery occlusion model in mice, which features platelet and VWF (von Willebrand Factor)-rich thrombi that reproduce r-tPA-resistant occlusion. Treatments were administered intravenously 1 hour after ferric chloride application by bolus injection or bolus followed by infusion, as translationally applicable. Laser speckle contrast imaging measured early reperfusion over the hour following treatment, and magnetic resonance imaging measured cerebral blood flow and lesion volume at 24 hours. Reperfusion 1 hour after treatment was greatest in caADAMTS13-treated animals. Later cerebral blood flow, 24 hours post-treatment, within the stroke-affected hypoperfused area was higher in caADAMTS13 and r-tPA but not TNK-treated mice. Functionally, this led to the absence of an initial behavioral deficit in caADAMTS13-treated mice, alongside a smaller lesion volume at 24 hours and reduced extent of bleeding. These findings demonstrate an overall suggestion that caADAMTS13 has improved thrombolytic efficacy, compared with current stroke treatments, against platelet-rich thrombi, for which there is currently an unmet clinical need.

Appropriate platelet function determines both the perioperative haemostasis and the risk of postoperative thrombotic complications in patients undergoing branched endovascular repair (bEVAR) of thoracoabdominal aortic aneurysm (TAAA). We aimed to assess the effect of bEVAR on platelet function and the predictive value of preoperative platelet function for postoperative bleeding. We measured platelet function using impedance aggregometry and total thrombus-formation analysis system in 50 consecutive patients, with TAAA undergoing elective bEVAR. After bEVAR, platelet reactivity was assessed using ASPI test, ADP test and TRAP test and thrombus size decreased, whereas time to clot formation increased, compared to baseline (p ≤ .042 for all). Preoperative platelet reactivity in the TRAP test was lower in patients who experienced post-operative bleeding, defined as ≥3 red blood cell units transfusion, compared to those who did not (p = .038). Baseline hemoglobin level <13 g/dl and TRAP test result ≤29.5 AUC increased the odds of bleeding by 5.4-fold and 6.8-fold, respectively, independent of other clinical variables. We conclude that in patients with TAAA undergoing bEVAR, platelet reactivity and platelet-rich thrombus formation decreased directly after the operation. Preoperative hemoglobin level and platelet reactivity in the TRAP test were independent predictors of postoperative bleeding complications.

Usefulness of serial in vivo imaging to directly assess the role of inflammation in thrombus resolution and organization.

Impact of antiplatelet therapy on hemostatic plug formation in the setting of thrombocytopenia.

Haemostasis refers to the physiological process aimed at repairing vessel injury and preventing bleeding. It involves four interlinked stages culminating in the formation of a platelet-fibrin haemostatic plug that is eventually dissolved once the vessel heals. In contrast, arterial thrombosis is a pathological condition resulting from atheroma exposure, triggering the formation of a platelet-rich thrombus that may obstruct blood flow, leading to the clinical manifestations of ischaemic cardiovascular disease. The following review will provide a comprehensive overview of the finely regulated endogenous antithrombotic mechanisms responsible for maintaining the haemostatic balance and preventing intravascular thrombosis. Thereafter, it will further detail the different stages and mechanisms governing the intricate interplay between the vessel, platelets, and the coagulation cascade in haemostasis, highlighting the most recent advances in platelet biology and function, to further elucidate the differential traits and players contributing to pathological arterial thrombus growth. The review will also delve into the impact of emerging cardiovascular risk factors on tilting the haemostatic balance towards a pro-thrombotic state, thereby increasing the patient's vulnerability to thrombotic events. Finally, it will underscore the importance of early screening for subclinical atherosclerosis through advanced imaging technologies capable of quantifying plaque burden and metabolic activity since they may set the stage for an increased thrombotic risk. Implementing proactive interventions to halt atherosclerosis progression or inducing its regression at early stages is crucial for preserving haemostasis and reducing the likelihood of ischaemic atherothrombotic disease.

Effectiveness of Adjunctive Caplacizumab Treatment in Immune Thrombotic Thrombocytopenic Purpura in Real-Life Setting. Retrospective Monocentric Cohort

Antiphospholipid Syndrome (APS) Is a Platelet Factor 4 (PF4)-Centric Immunothrombotic Disorder

Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds from time to time. In hemostasis, self-limiting platelet aggregation leads to the formation of a structured thrombus in which bleeding cessation comes from capping the hole from the outside. Detailed characterization of this structure could lead to distinctions between hemostasis and thrombosis, a case of excessive platelet aggregation leading to occlusive clotting. An imaging-based approach to puncture wound thrombus structure is presented here that draws upon the ability of thin-section electron microscopy to visualize the interior of hemostatic thrombi. The most basic step in any imaging-based experimental protocol is good sample preparation. The protocol provides detailed procedures for preparing puncture wounds and platelet-rich thrombi in mice for subsequent electron microscopy. A detailed procedure is given for in situ fixation of the forming puncture wound thrombus and its subsequent processing for staining and embedding for electron microscopy. Electron microscopy is presented as the end imaging technique because of its ability, when combined with sequential sectioning, to visualize the details of the thrombus interior at high resolution. As an imaging method, electron microscopy gives unbiased sampling and an experimental output that scales from nanometer to millimeters in 2 or 3 dimensions. Appropriate freeware electron microscopy software is cited that will support wide-area electron microscopy in which hundreds of frames can be blended to give nanometer-scale imaging of entire puncture wound thrombi cross-sections. Hence, any subregion of the image file can be placed easily into the context of the full cross-section.

Background: Red blood cells (RBC)-rich thrombus are more easily retrieved via endovascular procedures while platelet-rich thrombus are more resistant to recanalization. Our aim was to generate a radiomics model able to identify both RBC and platelet-rich thrombus at CT admission in patients undergoing mechanical thrombectomy. Methods: We included consecutive patients that received mechanical thrombectomy due to a large vessel occlusion in which thrombi was obtained. Thrombi obtained during the procedure were hematoxiline-eosine processed and proportions of RBC were determined. Relative proportion of the platelets in the thrombi was quantified by using a immunohistochemical staining recognizing CD61. We considered RBC-rich thrombi those with a content of RBC&gt;30% and platelet-rich thrombi those with a content of CD61&gt;70%. Thrombi were segmented manually on co-registered non-contrast CT (NCCT) and CT angiography (CTA) at admission (&lt;1mm thickness). 804 quantitative radiological features were extracted from the NCCT and CTA images. The most relevant features were selected with a genetic algorithm. Xgboost models were trained and one-versus-all cross-validated on the selected features to classify RBC and platelet-rich thrombi. Results: From 97 patients with histologically and radiologically available thrombi, 43% were female and mean age was 71(±12) years. The area under the curve for detecting RBC&gt;30% was 0.938 (sensitivity 86%, specificity 86%, PPV 84%). Regarding platelet-rich thrombus, the area under the curve for detecting CD61&gt;70% was 0.89 (sensitivity 83%, specificity 84%, PPV 74%). Feature importance by imaging type for RBC-rich and platelet rich thrombus was 70.33% and 69.8% for NCCT and 29.67% and 30% for CTA, respectively. The most important feature types for RBC-rich thrombus were histograms (51%) and first order (27%) while the most important features for platelet rich-thrombus were histograms (94%) followed by texture (5.94%). No information about shape improved any model. Conclusion: Our radiomics model can reliably identify RBC and platelet-rich thrombi. Fast identification of thrombus histological components on CT at arrival can help to design the preferred therapeutic strategy

Ultrasound (US)-enhanced microbubble (MB) therapy has been investigated as a therapeutic technique to facilitate the thrombolysis for the treatment of pericardial and microvascular obstruction. This study sought to assess the therapeutic effects of long-pulsed US-assisted MB-mediated recombinant tissue plasminogen activator (rt-PA) thrombolysis in a rat model of platelet-rich thrombus. Ferric chloride (10%) was used to induce total arterial occlusion before formation of platelet-rich thrombi. Therapeutic long-tone-burst US (1 MHz, 0.6 MPa, 1,000-µs pulse length) was used, and 2.9×109/mL of lipid MBs and 1 mg/mL of rt-PA were infused. Subsequently, 42 Sprague-Dawley (SD) male rats were randomly divided into seven groups: (I) control; (II) rt-PA; (III) high duty cycle US + MB; (IV) low duty cycle US + rt-PA; (V) high duty cycle US + rt-PA; (VI) low duty cycle US + rt-PA + MB; and (VII) high duty cycle US + rt-PA + MB. The recanalization grades were evaluated after 20 minutes' treatment. Compared to the control, there was significant improvement in recanalization in the US + rt-PA groups (P=0.01 vs. control), US (low duty cycle) + rt-PA + MB (P=0.003 vs. control) and US (high duty cycle) + rt-PA + MB (P<0.001 vs. control) groups, in which recanalization was successfully achieved in all rats. Long-pulsed US-enhanced MB-mediated rt-PA thrombolysis offered a powerful approach in the treatment of platelet-rich thrombus.

Pathogen Reduced Cryoprecipitated Fibrinogen Complex (IFC) and Cryoprecipitated AHF Contain Von Willebrand Factor with Comparable Binding to Collagen and Support Shear-Induced Platelet Thrombus Formation

PB1508 Massive Platelet-Rich Thrombus Formation in Small Pulmonary Vessels in Amniotic Fluid Embolism

H2O2-triggered "off/on signal" nanoparticles target P-selectin for the non-invasive and contrast-enhanced theranostics for arterial thrombosis.

Thrombotic thrombocytopenic purpura (TTP) is a rare and life-threatening thrombotic microangiopathy characterized by microangiopathic hemolytic anemia, severe thrombocytopenia, and organ ischemia associated with disseminated microvascular platelet-rich thrombus. Before the introduction of plasma therapy, acute TTP was almost universally fatal, which improved survival from < 10 to 80-90%. However, patients who survived an acute attack were at high risk for recurrence and long-term morbidity. It was reported that daratumumab can eradicate persistent ADAMTS13-inhibiting autoantibodies and restore ADAMTS13 activity in two patients with relapsed immune-mediated TTP without associated adverse drug reactions. Here we report a case series of patients with initial diagnosed acquired TTP treated with combination regimens containing daratumumab. All the patients achieved clinical response after the initial treatment. Three patients achieved clinical remission, one patient relapsed and one patient suffered an exacerbation during follow-up. The two patients were retreated with glucocorticoids, plasma exchange combined with daratumumab, and clinical remission was achieved again. Combination of daratumumab in the treatment of initial diagnosed acquired thrombotic thrombocytopenic purpura can rapidly restore ADAMST13 activity and turn negative for ADAMST13 inhibitors, resulting in long-term remission in patients.