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3750 Theme and Variation: Structuring Thrombus Formation from Jugular/Arterial Puncture Wounds to Occlusive Clots in a Mouse Model

Program: Oral and Poster Abstracts
Session: 301. Vasculature, Endothelium, Thrombosis and Platelets: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Biological Processes
Monday, December 12, 2022, 6:00 PM-8:00 PM

Brian Storrie, PhD1, Irina D Pokrovskaya, MS2*, Michael W. Webb, Ph.D.3*, Smita Joshi, Ph.D.4*, Sidney W Whiteheart, PhD5, Maria Aronova, PhD6* and Richard D. Leapman7*

1Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences, Cleveland, OH
2Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences, Little Rock, AR
3Physiology and Biophysics, University of Arkansas Med Sci, Little Rock, AR
4Department of Biochemistry, University of Kentucky, Lexingtton, KY
5Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY
6Laboratory for Cellular Imaging and Molecular Biophysics, NIH, NIBIB, Bethesda, MD
7National Institute of Biomedical Imaging aand Bioengineering, National Institutes of Health, Bethesda, MD

Hemostasis in veins and arteries has been assumed to be different processes, e.g., white vs. red clots. We took the unbiased experimental approaches of 3D EM across full clot volumes and high-resolution heat mapping of platelet activation state to probe for commonalities and variations using controlled mouse models. Remarkably, the thrombi formed, 5 min post-puncture, in the jugular vein and femoral artery showed considerable consistency in organization. There were vaulted cavities within the puncture wound and apparent extravascular capping of the puncture hole to produce bleeding cessation. Notable differences in the venous thrombi were a greater activation of platelets lining the surfaces of the vaulted columns, an outcome that we attribute to lowr flow rates resulting in the longer retention of activating signals, and in the “hanging drop” nature of the arterial thrombi anchored to the extrema with an outward, extravascular bulge of the thrombus. We attribute these variations to arterial vessel wall properties and higher flow rates. At 20 min post-puncture, the intravascular crown of the venous wound thrombi was enriched in loosely adherent platelets in a low activation state. Similarly, the outer portion of the intravascular arterial thrombus was rich in low activation platelets including obviously discoid platelets. Strikingly, the arterial thrombus was subject to rebleeding and ate 20 min displayed fractured extravascular features indicative of an unstable clot.

The puncture wound thrombi form in the relatively small volume of the immediate wound site. In contrast, occlusive clots fill the cylindrical volume of the vessel. To us, the overall structure of the wound thrombi suggested a predictive hypothesis for induced occlusive clotting. We propose that 1) the exposed intima lining the arterial vessel wall provides a cylindrical substratum for the generation of a tightly adherent platelet layer anchoring the forming clot and 2) the central volume of the clot resembles the intravascular crown of the puncture wound thrombus. Hence, the prediction of a central volume of the occlusive clot consisting of loosely adherent to discoid shaped platelets. That was result found for FeCl3 induced femoral clots by wide area-TEM (WA-TEM) i.e., a central volume rich in discoid shaped platelet and few red blood cells were trapped in the tightly adherent rim of the clot.. Using sequential block face SEM, we have begun to image and volume render carotid clots by sequential block face SEM. Analysis of 5,000 sequential images from the downstream half of the carotid clot, showed that the structure gradually tapered in a conical manner and contains few red blood cells. We propose that there are indeed organizational themes shared by the puncture wound thrombus in venous or arterial settings and the occlusive clot in which vascular damage is restricted to the endothelial cell layer.

Work was supported by NIH grants (BS) R01 HL155519 and P20 HL146373 (Joel Bennett, principal investigator), R35 HL150118 (SWW) and by intramural NIBIB research program (RDL).

An occlusive FeCl3 clot in the carotid artery of a C57BL/6 mouse imaged by sequential block face SEM over a length of 1.2 millimeters. A. cross section of the clot 200 microns in from the mid-occlusion starting point for sectioning. B. Zoom of A) to illustrate the low level of trapped red blood cells within the clot. C. cross section of the clot 900 microns in from the starting point showing how the clot has now tapered down and fills a minor portion of the artery. D. Rendering of 1.2 millimeters of the clot (blue), vessel wall (green) and in gray a single plane image. Flow was from left to right. Note that the clot progressively tapers to a point in the downstream direction.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH