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Whenever there are minor/major injury to blood vessels, the platelets, fibrin, thrombin, etc. are recruited. They then seal the wound and block bleeding.
What tells them that their job is done?
Exposed collagen at the site of vessel damage simultaneously initiates plates aggregation and the clotting cascade. So, when the wound is sealed and no more collagen is exposed, which is the main factor to activate the cascade, the blood clothing cascade stops.
What stops the coagulation process? - Biology
By the end of this section, you will be able to:
- Describe the three mechanisms involved in hemostasis
- Explain how the extrinsic and intrinsic coagulation pathways lead to the common pathway, and the coagulation factors involved in each
- Discuss disorders affecting hemostasis
Platelets are key players in hemostasis, the process by which the body seals a ruptured blood vessel and prevents further loss of blood. Although rupture of larger vessels usually requires medical intervention, hemostasis is quite effective in dealing with small, simple wounds. There are three steps to the process: vascular spasm, the formation of a platelet plug, and coagulation (blood clotting). Failure of any of these steps will result in hemorrhage—excessive bleeding.
Laboratory hemostasis: from biology to the bench
Physiological hemostasis is an intricate biological system, where procoagulant and anticoagulant forces interplay and preserves blood fluidity when blood vessels are intact, or trigger clot formation to prevent excessive bleeding when blood vessels are injured. The modern model of hemostasis is divided into two principal phases. The first, defined as primary hemostasis, involves the platelet-vessel interplay, whilst the second, defined as secondary hemostasis, mainly involves coagulation factors, damaged cells and platelet surfaces, where the so-called coagulation cascade rapidly develops. The activation and amplification of the coagulation cascade is finely modulated by the activity of several physiological inhibitors. Once bleeding has been efficiently stopped by blood clot formation, dissolution of the thrombus is essential to restore vessel permeability. This process, known as fibrinolysis, also develops through coordinate action of a vast array of proteins and enzymes. An accurate diagnosis of hemostasis disturbance entails a multifaceted approach, encompassing family and personal history of hemostatic disorders, accurate collection of clinical signs and symptoms, integrated with laboratory hemostasis testing. Regarding laboratory testing, a reasonable approach entails classifying hemostasis testing according to cost, complexity and available clinical information. Laboratory workout may hence initiate with some rapid and inexpensive "screening" tests, characterized by high negative predictive value, then followed by second- or third-line analyses, specifically aimed to clarify the nature and severity of bleeding or thrombotic phenotype. This article aims to provide a general overview of the hemostatic process, and to provide some general suggestions to optimally facilitate laboratory hemostasis testing.
Keywords: bleeding blood coagulation laboratory hemostasis platelets thrombosis.
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- The clotting process. World Federation of Hemophilia website. http://www.wfh.org/en/page.aspx?pid=635. Updated January 2014. August 21, 2019.
- Mackman N, Tilley RE, Key NS. Role of the extrinsic pathway of blood coagulation in hemostasis and thrombosis. Arteriosclerosis, Thrombosis, and Vascular Biology. 200727:1687-1693. https://www.ahajournals.org/doi/10.1161/ATVBAHA.107.141911. Published June 7, 2007. Accessed August 26, 2019.
- What are rare clotting factor deficiencies? World Federation of Hemophilia website. http://www1.wfh.org/publication/files/pdf-1337.pdf. Updated May 2014. Accessed August 21, 2019.
- Hemophilia. NIH Genetics Home Reference website. https://ghr.nlm.nih.gov/condition/hemophilia. Accessed September 3, 2019.
What stops the coagulation process? - Biology
Hemostasis is the natural process that stops blood loss when an injury occurs.
Explain the steps involved in hemostasis
- Hemostasis is the natural process that stops blood loss when an injury occurs.It involves three steps: (1) vascular spasm ( vasoconstriction ) (2) platelet plug formation and (3) coagulation.
- Vasoconstriction is a reflex in which blood vessels narrow to increase blood pressure.
- Next, platelet plug formation involves the activation, aggregation, and adherence of platelets into a plug that serves as a barrier against blood flow.
- Coagulation involves a complex cascade in which a fibrin mesh is cleaved from fibrinogen.
- Fibrin acts as a “molecular glue” during clot formation, holding the platelet plug together.
- hemostasis: The process of slowing and stopping the flow of blood to initiate wound healing.
- coagulation: The process by which blood forms gelatinous clots.
- heparin: A fibrinolytic molecule expressed on endothelial cells or produced as a blood thinner medicine. It prevents activation of platelets and clotting factors.
Hemostasis is the natural process in which blood flow slows and a clot forms to prevent blood loss during an injury, with hemo- meaning blood, and stasis meaning stopping. During hemostasis, blood changes from a fluid liquid to a gelatinous state.
Steps of Hemostasis
Hemostasis includes three steps that occur in a rapid sequence: (1) vascular spasm, or vasoconstriction, a brief and intense contraction of blood vessels (2) formation of a platelet plug and (3) blood clotting or coagulation, which reinforces the platelet plug with fibrin mesh that acts as a glue to hold the clot together. Once blood flow has ceased, tissue repair can begin.
Angiogenesis Generates New Blood Vessels: Blood vessel with an erythrocyte (red blood cell) within its lumen, endothelial cells forming its tunica intima or inner layer, and pericytes forming its tunica adventitia (outer layer).
Intact blood vessels are central to moderating blood’s clotting tendency. The endothelial cells of intact vessels prevent clotting by expressing a fibrinolytic heparin molecule and thrombomodulin, which prevents platelet aggregation and stops the coagulation cascade with nitric oxide and prostacyclin. When endothelial injury occurs, the endothelial cells stop secretion of coagulation and aggregation inhibitors and instead secrete von Willebrand factor, which causes platelet adherence during the initial formation of a clot. The vasoconstriction that occurs during hemostasis is a brief reflexive contraction that causes a decrease in blood flow to the area.
Platelet Plug Formation
Platelets create the “platelet plug” that forms almost directly after a blood vessel has been ruptured. Within twenty seconds of an injury in which the blood vessel’s epithelial wall is disrupted, coagulation is initiated. It takes approximately sixty seconds until the first fibrin strands begin to intersperse among the wound. After several minutes, the platelet plug is completely formed by fibrin.
Contrary to popular belief, clotting of a skin injury is not caused by exposure to air, but by platelets adhering to and being activated by collagen in the blood vessels’ endothelium. The activated platelets then release the contents of their granules, which contain a variety of substances that stimulate further platelet activation and enhance the hemostatic process.
When the lining of a blood vessel breaks and endothelial cells are damaged, revealing subendothelial collagen proteins from the extracellular matrix, thromboxane causes platelets to swell, grow filaments, and start clumping together, or aggregating. Von Willebrand factor causes them to adhere to each other and the walls of the vessel. This continues as more platelets congregate and undergo these same transformations. This process results in a platelet plug that seals the injured area. If the injury is small, the platelet plug may be able to form within several seconds.
If the platelet plug is not enough to stop the bleeding, the third stage of hemostasis begins: the formation of a blood clot. Platelets contain secretory granules. When they stick to the proteins in the vessel walls, they degranulate, thus releasing their products, which include ADP (adenosine diphosphate), serotonin, and thromboxane A2 (which activates other platelets).
First, blood changes from a liquid to a gel. At least 12 substances called clotting factors or tissue factors take part in a cascade of chemical reactions that eventually create a mesh of fibrin within the blood. Each of the clotting factors has a very specific function. Prothrombin, thrombin, and fibrinogen are the main factors involved in the outcome of the coagulation cascade. Prothrombin and fibrinogen are proteins that are produced and deposited in the blood by the liver.
When blood vessels are damaged, vessels and nearby platelets are stimulated to release a substance called prothrombin activator, which in turn activates the conversion of prothrombin, a plasma protein, into an enzyme called thrombin. This reaction requires calcium ions. Thrombin facilitates the conversion of a soluble plasma protein called fibrinogen into long, insoluble fibers or threads of the protein, fibrin. Fibrin threads wind around the platelet plug at the damaged area of the blood vessel, forming an interlocking network of fibers and a framework for the clot. This net of fibers traps and helps hold platelets, blood cells, and other molecules tight to the site of injury, functioning as the initial clot. This temporary fibrin clot can form in less than a minute and slows blood flow before platelets attach.
Next, platelets in the clot begin to shrink, tightening the clot and drawing together the vessel walls to initiate the process of wound healing. Usually, the whole process of clot formation and tightening takes less than a half hour.
Vasoconstriction: Microvessel showing an erythrocyte (E), a tunica intima of endothelial cells, and a tunica adventitia of pericytes.
What stops the coagulation process? - Biology
One of the most crucial steps of cheese making: coagulation is the step that transforms liquid milk into solid curd.
This step of the cheese making process is where the chemical magic is visible to the naked eye (and hand). Coagulation is the push-off-the-cliff that turns milk into cheese. Liquid milk is converted into a solid mass. This solid mass is often called “curd”, “gel” or the “coagulum”. Coagulation can occur in a few different ways: enzyme action, acid addition, or acid/heat addition. These three processes will be the foci of this post.
Coagulation of milk: before (left) and after (right)
Milk Chemistry Review
If you haven’t already, check out the previous post on milk chemistry. Specifically the section on protein. That information is crucial to understanding the rest of this post. As mentioned in the milk chemistry post, the protein of most interest in cheese making is casein. Casein micelles are covered with a negatively-charged “hairy” layer of κ-casein.
In milk, these casein micelles float around and bounce off each other. Those κ-casein hairs get in the way and prevent the casein from sticking and aggregating. Our goal in cheese making is to make those casein micelles stick together somehow. Once they stick together, a domino effect occurs, and eventually you form a mesh of casein micelles that form the structure/body of the cheese.
κ-casein hairs cause casein micelles to bounce off each other in milk
How we get those micelles to stick together is what coagulation is all about! The coagulation (or “clotting”) process is done to encourage those casein micelles to stick together somehow. Enzymes (rennets), acid, and acid/heat can all be used to encourage this process. The exact mechanism of each differs and will be discussed below.
Coagulation is getting those casein micelles to stick together
(This is an example of micelles after having the hairs clipped off)
Acid coagulated, acid-set, lactic curd, and lactic-set are all monikers that refer to using acid to coagulate milk. That acid can either be added directly or can be produced by starter cultures. A few examples of acid coagulated cheeses include cottage cheese, quark, and chèvre.
In this case, the goal is to neutralize the negative charge that is surrounding the casein micelles. In milk, all those negatively charged micelles bounce off each other due to them all having a negative charge. We often call caseins “polar” due to all this charge. Think about how magnets repel if you try to push together two of the same poles. Adding acid, in effect, is like adding positive charge. The addition of acid neutralizes the micelle surface and this allows them to bump into each other and stick. This effect is the most prominent at the isoelectric point of casein, pH = 4.6. If you recall from our last post about cheese texture, acid dissolves the calcium “glue” from the casein micelles. This means acid-set cheese is usually softer.
Acid neutralizes casein micelles and allows them to clot
Rennet coagulation refers to the addition of enzymes to milk in order to make it clot. Many cheeses fall into this category: cheddar, gouda, queso fresco, and many others.
Rennet enzymes act like a razor and shave off the κ-casein hairs. Without the hairs, the micelles can now stick, aggregate, and form the backbone of cheese structure. An interesting property of enzymes is that they are re-used in chemical reactions. This means a little bit of rennet goes a long way in the coagulation process. At home, a ¼ to ½ teaspoon is usually sufficient to clot 2 gallons of milk. The aggregation occurs when about 80-90% of the κ-casein hairs are clipped off. Not shown in the picture below is the required calcium that acts as a "glue" between the caseins.
Rennet clips off the hairy layer and allows the casein micelles to attach
Acid & Heat Coagulation
The last regime of coagulation on the docket could probably be considered a subset of acid coagulation. In this process, acid and heat are used to clot milk. Examples of this include ricotta, mascarpone, and paneer.
We’ve already discussed how acid affects casein, but what about heat? In this case, heat affects the other main type of milk protein we haven’t discussed yet, whey. Whey proteins are denatured (unraveled) by heat exposing “sticky” portions of their structure. These sticky ends can bond to each other across whey proteins or bond to casein proteins. Acid can now also contribute to whey coagulation now that they have been denatured by heat. The result is a matrix of coagulated whey protein, and if casein is present, a matrix of coagulated whey/casein.
Heat causes whey proteins to participate in the coagulation fun!
(Not to scale, like every other diagram on the site)
An Overview of the Blood Clotting Process
The blood clotting process is complex and involves many reactions. However, the process can be summarized in three steps.
- A complex known as a prothrombin activator is produced by a long sequence of chemical reactions.
- The prothrombin activator converts a blood protein called prothrombin into another protein called thrombin.
- Thrombin converts a soluble blood protein called fibrinogen into an insoluble protein called fibrin.
- Fibrin exists as solid fibres which form a tight mesh over the wound. The mesh traps platelets and other blood cells and forms the blood clot.
Prothrombin and fibrinogen are always present in our blood, but they aren&apost activated until a prothrombin activator is made when we&aposre injured.
Pineapple (Ananas comosus)
Bromelain’s Effect on Blood Clotting
The intrinsic and extrinsic pathways of blood coagulation play a vital role in preventing blood loss by forming clots. During certain pathological conditions these clotting factors can cause serious problems to individuals and lead to deadly diseases like thrombosis and embolism. Drugs that help in breaking down these clots are called anticoagulants. The anticoagulating effect of bromelain obtained from pineapple was first identified in 1972 when it showed an anticoagulant effect in 17 out of 20 volunteers after oral administration. This was further studied in animal models with bromelain found to be very effective. Bromelain plays an active role in blood fibrinolytic activity, decreasing the concentration of active fibrin which is a protein involved in clotting. At elevated concentrations bromelain increased activated partial thromboplastin time and prothrombin time. When investigating the mechanism of action, bromelain was found to be active in fibrinolysis—activating the conversion of plasminogen to plasmin which plays a role in degrading fibrin ( Castell et al., 1997 Lorkowski, 2012 Seifert et al., 1979 Winter, 1990 ).
How it all starts: Initiation of the clotting cascade
The plasma coagulation system in mammalian blood consists of a cascade of enzyme activation events in which serine proteases activate the proteins (proenzymes and procofactors) in the next step of the cascade via limited proteolysis. The ultimate outcome is the polymerization of fibrin and the activation of platelets, leading to a blood clot. This process is protective, as it prevents excessive blood loss following injury (normal hemostasis). Unfortunately, the blood clotting system can also lead to unwanted blood clots inside blood vessels (pathologic thrombosis), which is a leading cause of disability and death in the developed world. There are two main mechanisms for triggering the blood clotting, termed the tissue factor pathway and the contact pathway. Only one of these pathways (the tissue factor pathway) functions in normal hemostasis. Both pathways, however, are thought to contribute to thrombosis. An emerging concept is that the contact pathway functions in host pathogen defenses. This review focuses on how the initiation phase of the blood clotting cascade is regulated in both pathways, with a discussion of the contributions of these pathways to hemostasis versus thrombosis.
Keywords: Blood coagulation contact pathway factor VII factor XII polyphosphate tissue factor.
Overview of the blood clotting…
Overview of the blood clotting cascade. The plasma clotting system is initiated in…
Stop the clots, spare the coagulation
(BOSTON) — One of the most important and fraught processes in the human body is inflammation. Inflammatory responses to injury or disease are crucial for recruiting the immune system to help the body heal, but inflammation can also cause dangerous blood clots and other conditions by inducing an overproduction of the coagulant protein thrombin. Activated protein C (APC), a naturally occurring anti-coagulant protein with anti-inflammatory and other protective effects, is used medically to treat severe blood infections and wounds by reducing inflammation of the endothelial cells that line blood vessels. However, its use is limited because it can inhibit thrombin too much, which impacts the blood’s ability to clot normally and increases bleeding risk.
The blood-vessel-on-a-chip consists of parallel channels (red) lined with human endothelial cells, through which whole blood and other compounds can be perfused, to mimic the function of blood vessels in the human body. Credit: Wyss Institute at Harvard University
Now, a collaborative team of researchers from the Division of Hemostasis and Thrombosis at Beth Israel Deaconess Medical Center (BIDMC) and the Wyss Institute at Harvard University have discovered that synthetic APC-mimicking small molecules called “parmodulins” provide anti-inflammatory and anti-thrombotic protection to endothelial cells on par with APC’s without interfering with normal blood clotting and coagulation, making them attractive new drug candidates. This work was enabled by leveraging the Wyss Institute’s Organ-on-a-Chip technology to model thrombosis within a human blood vessel in vitro. The results are reported in this week’s issue of Proceedings of the National Academy of Sciences.
“We essentially performed a mini pre-clinical trial of parmodulins’ effect on the endothelium, and not only determined the pathway through which parmodulins function, but also demonstrated that they help protect endothelial cells from inflammatory damage,” says former Wyss postdoc Abhishek Jain, Ph.D., who is now an Assistant Professor and director of the Bioinspired Translational Microsystems lab at Texas A&M University.
The target protein on which both APC and parmodulins act is the transmembrane protein protease-activated receptor 1 (PAR1), which is present on both endothelial cells and platelets that circulate through the blood and promote clotting, making mechanistic analysis difficult. PAR1 was originally identified as a receptor for thrombin, which is a crucial part of the inflammatory process. However, when PAR1 is activated by APC on endothelium, it triggers anti-inflammatory, anti-apoptotic, and barrier-fortifying pathways, all of which help protect cells from the negative effects of inflammation.
In addition to activating PAR1, APC also independently inhibits the generation of thrombin, which is an essential component of healthy blood clotting – but inhibiting thrombin too much leads to uncontrolled bleeding. Knowing that parmodulins bind to PAR1, the team of scientists and clinicians set out to find a way to activate endothelial PAR1 and reduce thrombic responses without thinning the blood, and thus provide a better alternative to APC.
To evaluate the activity of parmodulins on endothelium, Karen De Ceunynck, Ph.D., postdoctoral research fellow at BIDMC and first author of the paper, incubated human endothelial cells with parmodulin 2 in vitro for 4 hours and then exposed them to the thrombin-inducing inflammatory agents lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α). In the parmodulin-exposed cells, both agents’ ability to generate thrombin was reduced by over 50% compared with non-parmodulin-exposed cells. However, parmodulin 2 did not inhibit the activity of factor V or factor X, proteins that function in blood coagulation. “We were intrigued by the notion that parmodulin 2 inhibited LPS- and TNF-mediated prothrombotic effects on the endothelial surface without impairing blood clotting” says De Ceunynck.
To confirm this theory, the team used a Wyss-developed blood-vessel-on-a-chip consisting of microfluidic channels embedded in a clear polymer chip, coated with collagen, and lined by human endothelial cells. Whole blood was perfused through the chip to simulate the flow conditions within human blood vessels, to which were added different pro- and anti-inflammatory compounds to evaluate the response of the endothelium.
Blood vessels exposed to the pro-inflammatory molecule LPS (third row) developed significant blood clots, while those pre-treated with parmodulin 2 (fourth row) and then exposed displayed a amount of coagulation similar to normal conditions (first row). Credit: Karen De Ceunynck, BIDMC
When the endothelial cells were exposed to TNF-α before being perfused with whole blood, platelets accumulated on the endothelium in a typical inflammatory response if the cells were first exposed to parmodulin 2 and then TNF-α, platelet accumulation was inhibited and the endothelium resumed its normal function. These results indicated that parmodulin exposure blocks the thrombotic response of endothelium to inflammatory stimuli without affecting blood coagulation in humans – a significant improvement over APC.
A series of tests in vitro performed by co-first author Christian Peters, Ph.D. at BIDMC, confirmed that parmodulin 2’s activation of PAR1 also induces cytoprotective responses in endothelial cells by inhibiting apoptosis (programmed cell death) induced by thrombin, TNF-α, and the apoptotic alkaloid staurosporine through a signaling pathway that begins with parmodulin 2’s binding to a specific site on the cytoplasmic side of PAR1. “We observed that the cytoprotective response induced by parmodulin 2 happened very quickly, and confirmed its rapid onset in time course and gene expression assays,” says Peters.
Additionally, in vivo studies in mice showed that parmodulin 2 reduces the binding of white blood cells to blood vessels and impairs platelet and fibrin accumulation at injury sites during the inflammatory response, confirming the anti-thrombotic and anti-coagulant activity of parmodulin 2 observed in vitro. Additionally, parmodulins do not interact with many of APC’s other binding partners, making it much more targeted to PAR1 and reducing other side effects.
“The discovery of an anti-inflammatory molecule that prevents endothelial thrombosis but also preserves normal blood coagulation is a major step toward an alternative and better approach to treating inflammatory disease,” says Rob Flaumenhaft, M.D., Ph.D., Professor of Medicine at Harvard Medical School, Chief of the Division of Hemostasis and Thrombosis at BIDMC, and corresponding author of the paper. “Furthermore, nearly all other pharmaceuticals that target transmembrane PAR1-like receptors bind to the exterior side of the receptor parmodulin 2 represents a paradigm shift for compounds targeting these receptors because it acts on the cellular side of the protein. We are excited to see if we can advance it to clinical trials.”
“This work provides another example of how organ-on-a-chip technology can enable faster and safer development and evaluation of drugs that could help patients around the world,” says co-author and Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at HMS and the Vascular Biology Program at Boston Children’s Hospital, as well as Professor of Bioengineering at Harvard’s John A. Paulson School of Engineering and Applied Sciences (SEAS).
Additional authors of the paper include Sarah Higgins, Ph.D., also a Research Fellow at BIDMC Omozuanvbo Aisiku, Ph.D, former Postdoctoral Research Fellow in the Division of Hemostasis and Thrombosis at BIDMC and currently a scientist at Instrumentation Laboratory Jennifer Fitch-Tewfik, Ph.D., former Postdoctoral Research Fellow in the Division of Hemostasis and Thrombosis at BIDMC and currently a teacher at Southeastern Regional Vocational Technical High School Sharjeel Chaudhry, a Predoctoral Fellow in the Division of Hemostasis and Thrombosis at BIDMC Chris Dockendorff, Ph.D., Assistant Professor at Marquette University and Samir Parikh, M.D., Associate Professor at HMS.
This research was supported by the National Heart, Lung, and Blood Institute and the Wyss Institute for Biologically Inspired Engineering at Harvard University.