The Hemostasis and Coagulation Page

Hemostasis:  The word hemostasis literally means, "stop the bleeding" or "prevent blood loss."

There are several mechanisms involved in hemostasis and, here, we divide the mechanisms into 4 general categories:

Part 1 of Hemostasis) Vascular spasm (also referred to as "vascular response"):  Vascular "spasm" refers to a local vasoconstrictive mechanism.  That is, trauma to the vessel wall causes vasoconstriction in both directions along the traumatized vessel.  The region of vasoconstriction of the traumatized vessel extends several centimeters in each direction from the area of trauma!  Vascular spasm reduces blood flow through the traumatized vessel and, thus, reduces blood loss.  Also, keep in mind that a number of factors that lead to coagulation (clotting) are released from the traumatized blood vessel wall.  Reducing blood flow through the traumatized vessel wall reduces the dilution of the clotting factors in the traumatized area.  If the factors were diluted, coagulation time would be increased (it would take longer to stop bleeding)!!!

A stimulus (trauma) from within the wall of the traumatized vessel causes the smooth muscle of the wall to constrict, reducing blood flow (loss) from the ruptured vessel.  Local vasoconstriction results partly from nervous reflexes (nociceptive) but mostly from local myogenic spasm.  The direct damage to the vessel wall causes action potential transmission and contraction through the vascular smooth muscle for several centimeters in each direction.  The sharper the cut, the greater the blood loss as less tissue is traumatized in a clean cut than in a rough or serrated cut or in a crushing wound.  The local vascular myogenic response (spasm) can last for hours; this restricts blood flow, reducing blood loss but also reducing dilution of the clotting factors during clot formation.

Of course, we see vascular spasm only in those blood vessels with smooth muscle, which means the arteries, veins, and arterioles.  Venules have very limited amounts of smooth muscle at their venous end, and capillaries have no smooth muscle at all... so vascular spasm does not work for venules or capillaries.  Vascular spasm is most effective in the arterioles, because they are small enough, but with enough muscle to nearly or completely close.  To the left, Angie Dickinson shaves Dean Martin with a straight razor in Rio Bravo while John Wayne laughs in the background - 1959!  (potential for a low trauma injury by a very sharp razor which would lead to a lot of bleeding - slow to clot because the sharp razor cuts through the tissue smoothly, causing little tissue damage!)

It may be hard to get a lot of information about crushing injuries because most of the time, crushing injuries are associated with death.

Part 2 of Hemostasis) Platelet plug formation (platelet response/reaction):  Here, platelets respond to factors released by the traumatized vessel wall in an attempt to quickly seal holes in the damaged vessel.  Platelet plug formation is important in all vessel types!

A nick in a blood vessel results in exposure of what we call "the wetable surface."  The wetable surface consists of injured endothelial cells (remember, endothelium lines the lumen of the vessels) and exposed collagen where platelets will stick.  The "platelet reaction" really consists of platelets sticking to the wetable surface, swelling and attracting other platelets.  When punctures in the blood vessels are small enough, platelet plugs will stop the blood flow and no blood clot is required.  Platelets contain contractile proteins (including actin and myosin), residual endoplasmic reticulum (Ca2+) and Golgi bodies; this means that platelets can synthesize ATP, prostaglandins, fibrin-stabilizing factor and endothelial growth factor.

Why do platelets stick to injured tissue?

a) The surface of platelets are covered with glycoproteins, so platelets stick to the rough surface provided by the injured endothelial cells or exposed collagen.

b) Both the smooth vascular endothelial surface and the outer membrane surface of platelets are negatively charged.  Remember, "like" charges repel one another, so platelets don't want to stick to the healthy endothelium that lines the blood vessels.  However, the damaged tissue that is exposed when the lining of a blood vessel is damaged is positively charged!  This positively charged, wetable surface will attract and hold the negatively charged platelet membranes.... via charge bonding!!!

When platelets contact damaged tissue inside a blood vessel wall, the damaged tissue triggers a response from the platelets called the "platelet reaction."  The platelets swell up and become increasingly sticky due to the release of glycoproteins.  The platelets begin to release a bunch of arachidonic acid metabolites (arachidonic is a fatty acid!).  Among the arachadonic acid metabolites released by platelets are thromboxanes (TX's) and prostaglandins (PG's).  Activated platelets also release serotonin (5-HT) and thrombopoietin!

But what do all of these factors released by platelets actually do, you ask??? Perhaps most importantly, these factors i) attract more platelets into the region of the damaged tissue, ii) cause vasoconstriction, and iii) increase the production rate of new platelets.  The reasons that we want to do all this may seem self-evident, but let's go into a bit more detail on these factors released by platelets.

a) Thromboxanes (TX's) and progstaglandins (PG's): These factors attract (positive chemotaxis) and activate surrounding platelets, causing platelets to clump together and form a loose platelet plug.  These arachidonic acid metabolites also induce vascular spasm!  In addition, these arachadonic acid metabolites cause platelets to release an ADH-like peptide (yes, ADH)!  This ADH-like peptide, released by the platelets, causes the platelets, themselves, to send out pseudopod-like processes to grab fibrin "threads." (this is an "autocrine" signal).  Platelets actually have specific fibrin binding sites and they grab fibrin to anchor blood clots together (more on this below)!

b) Serotonin (5-HT) induces vascular spasm!  You can read more about vascular spasm below!

c) Thrombopoietin stimulates the production of more platelets!

People develop small tears in hundreds of vessels every day, but the holes are small and are routinely plugged by platelets.  In thrombocytopenia, these routine vascular tears are not repaired and petechial hemmorhages will be seen under the skin, in addition to internal hemorrhages which cannot be seen.  Formation of "platelet plugs" depends on TX's and PG's, and may be inhibited by NSAID's (non-steroidal anti-inflammatory drugs, eg. aspirin, indomethacin; these anti-inflammatory drugs suppress cyclo-oxygenase production of PG's or lipoxygenase production of TX's).  The platelet mechanism does not interfere with vessel function, whereas full coagulation sometimes does; that is, blood clots may actually block blood flow by blocking the lumen of some small blood vessels.

Part 3 of Hemostasis) Blood coagulation (clotting):  One way to think of a clot is as follows: the platelet reaction gets going and a network of fibrin molecules are added to link the platelets together and make a stronger patchwork over a traumatized area.  In fact, platelets appear to have specific binding sites for attachment to fibrin.  Fibrin "threads" are strong protein filaments!  As the "patchwork" or "clot" is formed, a bunch of blood cells get trapped in the middle of the "net."  This picture shows a blood clot and you can clearly see the enormous number of RBC's trapped within the fibrin network!  Remember, the RBC's are not really part of the clotting mechanism, they simply get caught up in the fibrin network and end up being used as "building blocks" in the plug that stops the bleeding!

We have a fairly simple construct (outline) of the factors involved in the clotting mechanisms (pathways).  Note that, as there are over 40 known endogenous pro-coagulants and anticoagulants, all known factors and cofactors in the pathways are not shown in this construct!

Under normal circumstances, anticoagulant activity outweighs pro-coagulant activity in the bloodstream and the blood keeps flowing.  When tissues are traumatized, pro-coagulant activity outweighs anticoagulant activity in the area of trauma in order to stop the bleeding.

Initiation of coagulation via the EXTRINSIC CLOTTING PATHWAY (the fast pathway):

The terms Extrinsic and Intrinsic can be more than a little bit confusing!  Extrinsic means "from outside the blood."  In terms of the "extrinsic clotting pathway," this means that the clotting mechanism is initiated (prothrombin activation) by factors from the blood vessel rather than from the blood, itself!

In the extrinsic clotting pathway, Factor III released by the traumatized tissue (the wetable surface) activates a simple series of biochemical reactions involving factors produced by platelets.  Note that activation of the extrinsic pathway leads to the conversion of prothrombin to thrombin.  This is an important step!

Subsequent steps in the pathway will lead to the formation of a blood clot within 15-20 seconds in severe trauma and within 1-4 minutes with minor trauma.  For this reason, the extrinsic clotting pathway is often called the "fast pathway."  The extrinsic pathway is faster than the intrinsic pathway simply because the extrinsic pathway is shorter... meaning, the extrinsic pathway has fewer chemical steps than the intrinsic pathway.  We used to remember that the extrinsic pathway was the fast pathway by saying, "the extrinsic pathway is extra fast!"

Initiation of coagulation via the INTRINSIC CLOTTING PATHWAY (the slow pathway):

In the intrinsic pathway, prothrombin activation begins within the blood; that is, in this pathway, prothrombin activation is driven by platelets, which are formed elements of the blood!

The term "intrinsic pathway" refers to a series of biochemical reactions that lead to clotting, where the process is driven by factors within the blood... that is, the process is driven by platelets!  Notice that the factor that keeps the pathway going is factor XII from platelets.  Even though factor XII is released from platelets, notice that activation of factor XII requires contact with traumatized tissue!  Once the pathway is initiated, activated platelets (intrinsic as they are part of the blood) will continue to drive the reactions in the pathway until bleeding stops.  Note that activation of the intrinsic pathway leads to the conversion of prothrombin to thrombin.   JUST LIKE IN THE EXTRINSIC PATHWAY!

The platelets will continue to drive the reactions in the intrinsic pathway even after the wetable surface is covered!  This means that the intrinsic pathway will continue to run until the hole in the vessel wall is plugged up, well sealed and no longer leaking!

The intrinsic pathway is sometimes referred to as the "slow pathway," as clotting will not begin for 3-4 minutes, even with moderate to severe trauma!

the three major steps in coagulation (clotting)

Under ideal circumstances, clot formation is complete within 3-6 minutes and clot retraction (syneresis) begins within 30-60 minutes, releasing serum from the clot and pulling opposing sides of the wound together for a tighter seal.  In clot retraction, a clot shrinks and releases plasma trapped within the clot (like squeezing a sponge).

1) formation of prothrombin activator occurs in response to trauma; formation of prothrombin activator is the goal of both the extrinsic and intrinsic pathway;  working together, the extrinsic and intrinsic pathways amplify formation of prothrombin activator; this is the point at which the extrinsic and intrinsic pathways merge

2) prothrombin activator promotes conversion of prothrombin to thrombin

3) thrombin promotes conversion of fibrinogen to fibrin; fibrin threads enmesh platelets, red and white blood cells and plasma

Platelets, fibrin and clot retraction:  A blood clot is a meshwork of fibrin threads anchored to platelets and trapping blood cells, platelets and plasma.  Fibrin readily adheres to the wetable surface, grabs platelets and holds them together.  Fibrin threads form and attach to "fibrin binding sites" on platelets, forming a strong "plug."  Platelet pseudopods formed during the platelet response expose additional fibrin binding sites.  Platelets trapped in the clot induce retraction (syneresis).  Platelets release microtubular elements containing actin- & myosin-like proteins that are activated by Ca2+ and help contract the clot.  Essentially, the pseudopod-like projections of platelets "grab" fibrin "threads" and pull, compressing the clot!  This clot retraction process is known as "syneresis."

Blood Clot

Blood Clotting is Compromised in Liver Disease or Vit K Deficiency: Prothrombin and fibrinogen are plasma proteins which are important in clotting.  As these factors are produced in the liver, blood clotting is compromised (impaired) in liver disease.  Vitamin K is an essential factor in prothrombin production, so blood clotting is also compromised in Vitamin K deficiency.

limits to clot expansion

The clot itself (especially platelets) stimulates activation of many clotting factors in an attempt to extend itself, primarily because thrombin proteolytically cleaves other factors to their active form (thrombin activates prothrombin and Clotting Factors VIII, IX, X, XI & XII).  Because thrombin activates so many of the clotting factors, the successful formation of thrombin is very important in clotting!

Q: So, what finally stops the growth of this apparently menacing clot blob, preventing it from "clogging up" the vasculature??? (ie. why does the entire vascular system not turn into a giant clot when we get cut?):

A: As thrombin is carried into areas where there is no tissue damage, the blood flow is greater and thrombin does not get "caught" by traumatized surfaces.  As we move more than a few centimeters from the traumatized tissue, thrombin becomes too dilute to stimulate further clotting.  In other words, as thrombin is carried out of the area of vascular spasm and into areas of greater blood flow, thrombin becomes too dilute to stimulate further clotting.

 

Part 4 of Hemostasis) Permanent repair and "dissolution" of the clot (dissolution means breakdown or to dissolve, and is sometimes referred to as "fibrinolysis"):  Fibroblasts invade the clot leading to fibrous tissue growth into the clot, permanently closing the hole.  This fibrous tissue patch is simply scar tissue in the blood vessel wall!  As the hole in the blood vessel undergoes permanent repair, the clot is gradually dissolved away (the process known as dissolution) by an enzyme known as plasmin.  Plasmin is an activated enzyme within clots; plasmin dissolves fibrin!

Invasion of the clot by fibroblast cells begins within hours of trauma.  The process is promoted by "fibroblast growth factor" released by platelets.  Fibrous tissue development (scar tissue) is complete within 7-10 days or less.  We often see the final effects of dissolution of a clot when we see a scab gradually peel away from the surface of our body when we are cut!

How does plasmin get activated? Thrombin rapidly activates conversion of fibrinogen to fibrin to quickly form blood clots.  At the same time, thrombin combines with thrombomodulin (the thrombin-thrombomodulin complex) to slowly trigger conversion of plasminogen to plasmin and to slowly destroy clotting factor V.  These processes requires Vitamin K!  Activated plasmin slowly dissolves clots which are no longer of use.  As Clotting Factor V is destroyed, clotting stops!

Thrombomodulin is a "safeguard" molecule in the clotting process.  We just heard about thrombomodulin in the paragraph above; it combines with thrombin to activate plasmin which, in turn, dissolves clots that are no longer needed.  The thrombin-thrombomodulin complex also helps stop the clotting process by inactivating Clotting Factor V.  But thrombomodulin plays another very important role!  Thrombomodulin is a thrombin binding protein produced by normal cell surfaces.  That simply means that thrombomodulin binds thrombin!  Free thrombin that "gets away" from the area of trauma has the ability to trigger clotting!  It would not be desirable to induce clotting in areas other than the area of trauma!  Thrombomodulin binds thrombin which is floating freely in circulation such that free circulating thrombin cannot cause clotting in healthy tissues.

In the Arteries & Veins: holes are sealed by vascular spasm, coagulation, platelet plugs and can be assisted by mechanical compression to reduce blood flow through the traumatized vessels.

In the Arterioles & Venules: holes are sealed by vascular spasm and platelet plug formation.  During surgical procedures, hemostats may be used to crush tissue around holes in vessels or to crush together the walls of severed vessels.  A hemostat used to crush and hold closed the end of a moderate-sized or small blood vessel (ie. clamp it and leave it in place for several minutes) can effectively help the vessel to be sealed!  Remember, when we clamp and crush, we increase the amount of trauma to the vessel wall; in turn, this increases the release of factors inducing the platelet response and clotting!  Sometimes we use artificial surface media to promote coagulation (an example would be GelFoam); small pads of sterile, absorbable material treated with pro-coagulants are pressed against bleeding tissues and act as a surface to promote coagulation.  GelFoam is very effective if you don't keep pulling on it to see if blood has started to clot to it!!!

In the Capillaries:  Capillary endothelial swelling, precapillary sphincter contraction and platelet response are the major mechanisms used to achieve hemostasis in capillaries.  Remember, there is no smooth muscle in the capillary wall!  The clotting mechanism is in place but will tend to block off individual capillaries when activated.  Remember, RBC's tend to pass through capillaries in single file; capillaries are very narrow!

If precapillary sphincter contraction does not occur, petechial hemorrhages (purpuric hemorrhages) are seen.  In Ricket's (Vit C deficiency, scurvy), petechial hemorrhaging is associated with the breakdown of the basement endothelium.

Blood Clotting Disorders and General Terminology

Hemophilia is a genetically inherited clotting insufficiency.

In Hemophilia A, also known as classic hemophilia, there is an insufficiency of Factor VIII (antihemophilic factor).  This form of hemophilia represents approximately 85% of all known cases of hemophilia.  Factor VIII is unstable in stored blood and, for this reasons, when transfusions are performed on persons with Hemophilia A, relatively fresh blood must be used.

In Hemophilia B, there is an insufficiency of Factor IX (Christmas factor).  This form of hemophilia represents approximately 15% of all known cases of hemophilia.  Unlike Factor VIII, Factor IX is stable in storage and transfusion with stored blood is feasible!

In Hemophilia C, which is very rare (about 1 in 100,000 births), there is a deficiency of Factor XI, which is the precursor for thromboplastin.  Factor XI also occurs in the intrinsic pathway, but in addition, Factor XI promotes activation of Factors VII and X in the extrinsic pathway.  While Hemophilia C is considered the "least severe" form of hemophilia, there is clearly no "good" hemophilia.  Surgery poses a threat to patients with Hemophilia C.  Factor XI is released from platelets and traumatized vessels.

Notice that the factors which are insufficient in both major forms of hemophilia are both found in the intrinsic clotting pathway!  This demonstrates something really important!  The extrinsic clotting pathway is functional in hemophilia!  The extrinsic clotting pathway, alone, does not stimulate clotting strongly enough to provide adequate hemostatic protection; without treatment with exogenous clotting factors, the blood of someone with hemophilia will clot, but the process is extraordinarily slow and any moderate trauma becomes life threatening!  So the intrinsic and extrinsic pathways synergize to amplify their effect on thrombin production and clotting!

Hemophilia is associated with X-linked recessive genes which are expressed in 1 of every 10,000 males, and in almost no females.  A mother carrying the disorder on a single X chromosome transmits the disease to half of her male offspring and transmits the carrier state to half of her female offspring.

Although people use the phrase often enough, there is no such thing as "mild hemophilia."  Hemophilia carries with it an increased mortality rate and would disappear were it not for the apparent occurrence of fresh mutations.  Strangely enough, this means that humans, or perhaps some subpopulation of humans, must be somewhat susceptible to this mutation.  The effect of this is that, in one-third of new hemophilia cases, there is no previous history of hemophilia in the immediate family.

SOME RELATED TERMINOLOGY

Thrombus: a clot at an inappropriate site.

Embolism, thromboembolism: blockage of vessels by disengaged clots; for example, in arteriosclerosis, infection, trauma; notably in areas where blood flow is low, roughened vessel surfaces are maintained and stimulation of clotting factors is prolonged.

Embolism in a coronary artery may lead to a heart attack.

Embolism in the brain may lead to stroke.

Pulmonary emboli: clots trapped in the lungs.

Massive pulmonary embolism: blood flow is blocked and increasing pressure may lead to massive pulmonary edema; death may ensue within a very short span of time (minutes to hours); the prognosis is grave

Septicemic shock: bacterial endotoxins activate clotting in small but numerous blood vessels, decreasing O2 availability to the tissues and leading to exacerbation of the shock; a very slim chance of survival

Progressive shock: recall from Unit One (Body Water Compartments) that acids produced by inadequately perfused tissues induce coagulation in the blood vessels (as we saw in irreversible hypovolemic shock).

Some Anticoagulants of Note

1.    Coumarin: (products of coumarin include coumadin (dicoumarol), rodenticides, warfarin, brodifacoum, super-warfarin... and many other anticoagulants!):  Coumadins compete with Vitamin K for reactive sites on liver enzymes that are responsible for the production of clotting Factors II, VII, IX and X.  That means that Vitamin K can be used as an antidote to dicoumarol poisoning.

Dicoumarol poisoning is a condition in which a patient has received an excess of coumadin, or in which cattle are allowed to eat too much hay containing molded sweet yellow clover (melilot).  "Sweet clover disease" in cattle results from conversion of coumarin to dicoumarol when sweet clover molds.  Molded hay generally occurs if hay is baled prior to allowing the hay to adequately "cure" (dry), but may also occur if hay bales are stored in damp conditions (for example, directly on a dirt floor, or in a barn with a leaky roof).  Rolled hay stored in an open field does not usually "mold" if the hay has been adequately cured before baling, because the sun and wind tend to dry the hay quickly following rainfall, preventing molding, although some mold may appear on the bottom of rolled bales stored in a field.

Warfarin, which is commonly used in anticoagulant therapy (to prevent clots) is, simply put, rat poison!  Warfarin causes death in rats by internal bleeding; warfarin is contained in the food substance that comes in rat bait.  Warfarin, dicoumarol, and coumadin are, essentially, all the same thing.  Warfarin can be used to prevent clots but it has no effect on existing clots!

2.  Ca2+ chelators like ethylene diamine tetra-acetic acid (EDTA):  EDTA chelates calcium ions to form a soluble complex.  Factors such as EDTA, EGTA, oxalates (in vitro), sodium citrate, ammonium citrate or potassium citrate (in vivo or in vitro), act as Ca2+ chelators.  By binding up Ca2+, they prevent Ca2+-dependent activation of clotting factors.

3.  Hirudin is a blood anticoagulant normally produced in the salivary glands of the medicinal leech (Hirudo medicinalis).  This short polypeptide (65 amino acids) is a potent inhibitor of the blood coagulation factor, thrombin.  Remember how important thrombin is, it activates numerous clotting factors!  In addition, hirudin is only weakly immunogenic and so it is well tolerated.  In clinical trials, hirudin has proven to be very convenient to administer, with patients' clotting responses returning to normal quickly after withdrawal from the hirudin treatment.

4.  Heparin:  Heparin is a negatively charged proteoglycan (mucopolysaccharide) which opposes platelet sticking (in vivo or in vitro).  Heparin is considered the anticoagulant of choice for red cell morphology studies.  Heparin stops the formation of thrombin from prothrombin, therefore stopping formation of fibrin from fibrinogen.

5.    Factor IXa anticoagulants:  This is a group of recently discovered low molecular weight compounds which inhibit the intrinsic clotting pathway.  It is an exciting find as these anticoagulants are easily synthesized, well tolerated and easily polymerized (attached) to medical devices and hardware that will be temporarily or permanently implanted into the body.

Prothrombin Time (Clotting Time Test) using anticoagulants:

INR (the "international normalized ratio" compares prothrombin time to an international standard).  INR values are used to indicate the effectiveness of anticoagulant therapy in patients.  For example, say a patient was receiving warfarin to prevent clotting following a heart valve operation... an INR of 2.0 would indicate that their prothrombin time was double that of the normal time.  An INR of 2.0 would indicate that the anticoagulant therapy was working... by slowing down the clotting process!

"Prothrombin time" is performed by collecting a blood sample into an "oxalated" tube.  The oxalate chelates Ca2+ in the blood and prevents the blood from clotting.  To start the test, an excess of Ca2+ and tissue thromboplastin (thromboplastin is clotting Factor III) are added to the sample.  Addition of Ca2+ allows clotting to proceed and clotting is strongly promoted by the excess thromboplastin, which promotes conversion of prothrombin to thrombin, which in turn coverts fibrinogen to fibrin.  The clotting time is strongly accelerated and should approximate 12 seconds.

Clotbusters:  "Clotbusters" are different than anti-coagulants in that... anti-coagulants are intended to prevent clots before they occur... whereas clot busters are intended to break apart existing clots or embolisms formed by clots.  Clot busters work by activating plasmin... the same factor responsible for dissolution of clots.  Clot busters include streptokinase and prothrombin activating factor.

David Currie.
Copyright 2000. All rights reserved.
Revised: January 05, 2009